Structural and Energetic Factors of the Increased Thermal
Stability in a Genetically Engineered Escherichia coli
Adenylate Kinase*
Simona
Burlacu-Miron,
Véronique
Perrier
,
Anne-Marie
Gilles
,
Elisabeth
Pistotnik
, and
Constantin T.
Craescu§
From INSERM U350, Institut Curie-Recherche, 91405 Orsay Cedex,
France and
Laboratoire de Chimie Structurale des
Macromolécules (URA D1129), Institut Pasteur, 7524 Paris Cedex 15, France
 |
ABSTRACT |
Several variants of Escherichia coli
adenylate kinase, designed to bind a Zn2+ ion, were
produced by site-directed mutagenesis. The metal binding and enzymatic
properties of the engineered variants have been described (Perrier, V.,
Burlacu-Miron, S., Bourgeois, S., Surewicz, W. K., and Gilles,
A.-M. (1998) J. Biol. Chem. 273, 19097-19101). Here
we report the structural properties and stability changes in a 4-Cys
variant which binds a Zn2+ ion and has an increased thermal
stability. CD studies indicate a very similar secondary structure
content in the wild type and the engineered variant. NMR analysis
revealed that the topology of the parallel
-sheet, belonging to the
protein core, and of the peripheral antiparallel
-sheet are also
conserved. The small local changes observed in the neighborhood of the
substitution sites reflect a more compact state of the metal-binding
domain. The Zn2+-bound quadruple mutant shows an increased
thermal stability, reflected in a 9 °C increase of the
mid-temperature of the first cooperative unfolding step. Binding of a
bisubstrate analog
P1,P5-di(adenosine-5')-pentaphosphate
increases, by about 7 °C, the midpoint of this transition in both
wild type and modified variant. The NMR data suggest that the
peripheral domains involved in substrate binding unfold during the
first denaturation step. Urea denaturation experiments indicate an
increased resistance against chemical unfolding of the
Zn2+-binding variant. In contrast, the Gibbs free energy of
unfolding (at physiologically relevant conditions) of the quadruple
mutant is lower than that of the wild type.
 |
INTRODUCTION |
Adenylate kinases (AKs)1
represent a class of small (20-26 kDa) and abundant
phosphotransferases involved in the reversible transfer of the terminal
phosphate group from ATP to AMP. They contribute to the maintenance of
a constant level of cellular adenine nucleotides, necessary for
energetic metabolism and nucleic acid synthesis. From the structural
point of view (Fig. 1) AKs belong to the
/
class (a five-stranded
-sheet surrounded by several
-helices), characteristic for many nucleotide-binding proteins (1).
In addition to this protein core, they contain generally two smaller
domains involved in the substrate binding. One is composed by two
helices (
2 +
3) and contributes to the mononucleotide binding,
NMP domain closing over bound AMP (residues 30-59). The other is
involved in binding of ATP and, due to its large movement, closing over
the bound nucleotide, it was called LID domain. In the mitochondrial
and most of the bacterial variants of AK, the LID domain has 38 residues (residues 122-159 in AKe) while
smaller cytosolic enzymes have only an irregular loop of 11 residues
(2, 3). In the AKe (a "long" kinase) the LID
domain contains a small four-stranded antiparallel
-sheet (4). X-ray
structures of the enzyme in complex with various ligands revealed that
this domain has significantly different positions relative to the
protein core, depending on the nature and number of bound ligands.
Therefore, it was inferred that complexation (particularly with ATP)
induces a large scale rotation/translation movement from a remote
position into a close contact with the main part of the protein (3-6).
Solution studies, using resonance energy transfer measurements (7),
confirmed the existence of such hinge motion of the LID domain upon
binding of Ap5A, AMP, and ATP. Nevertheless, the short AKs
conserve their catalytic activity despite the absence of the LID
domain, but seems to loose the high specificity for ATP (8).

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Fig. 1.
Schematic representation of the global
structure of Ake drawn with MOLSCRIPT
software (22) and the atomic coordinates (4ake) determined by
Müller et al. (4). Some of the side chains
mentioned in the text are shown explicitly and labeled.
|
|
A screening of bacterial AKs has revealed that the enzymes from
Gram-positive organisms bind a Zn2+ ion via
coordinative bonds to four side chains (Cys and Asp) situated in the
-hairpin loops of the LID domain (9). Based on sequence comparison
with AK from the Gram-positive, thermophilic Bacillus
stearothermophilus, we have designed several variants of the
Escherichia coli enzyme involving three- or four-amino acid
substitutions in the LID domain, at positions His126,
Ser129, Asp146, and Thr149. In the
companion study (10) we showed that some of these variants bind a
Zn2+ ion and conserve the enzymatic activity. In this study
we report the results of NMR and CD experiments aimed to characterize
in more detail the structural consequences of the amino acid
substitutions and zinc binding. The 4-Cys variant has an increased
thermal and chemical (urea) stability, but the structural stability at
physiologically relevant conditions is slightly reduced.
 |
EXPERIMENTAL PROCEDURES |
All chemicals were of highest purity available. Protein design,
expression, and purification were described in our companion study
(10).
Circular Dichroism--
The far-UV CD spectra were recorded on a
Jasco 700 spectropolarimeter in a 0.1-cm quartz cells. The spectra were
obtained as an average of five runs and were corrected for the
contribution of the buffer. For the thermal denaturation experiments we
followed the ellipticity at 222 nm as a function of temperature,
programmed to rise uniformly at 60 °C/h.
Urea denaturation was monitored by the changes in ellipticity at 222 nm. Aliquots of concentrated solutions of protein were added to urea
solutions (0-8.8 M) and the samples were incubated until
equilibrium was reached at the temperature chosen for determining the
unfolding curve (20 °C). The Gibbs free energy of denaturation was
estimated by Equation 1
|
(Eq. 1)
|
where K is the equilibrium constant of denaturation
at a given reaction temperature (RT) and urea concentration. The
measured free energy difference is considered (11) a linear function of
the denaturant concentration
|
(Eq. 2)
|
where
G(H2O), the extrapolated value
of
G at zero molar denaturant, represents the intrinsic
stability of the protein in absence of urea.
NMR Methods--
Samples were prepared by dissolving the
lyophilized protein in potassium phosphate buffer (50 mM)
in 2H2O or in 1H2O
containing 7% 2H2O at pH 6.5. NMR spectra were
obtained on a Varian Unity 500 NMR spectrometer, using standard methods
for pure absorption DQF-COSY (12), NOESY (13), and TOCSY experiments
with 30-60-ms spin lock times (14). Proton chemical shifts were
referenced relative to the water signal which, in our conditions, at
308 K resonates at 4.69 ppm from the sodium
2,2-dimethyl-2-silapentane sulfonate signal.
 |
RESULTS |
Structure Characterization of the Engineered Variant--
CD
spectroscopy in the far-UV region is a sensitive measure of the
secondary structure content in polypeptides and is particularly useful
in comparing the backbone structural differences between related
proteins (15). The similarity between the CD spectra of the wild-type
and mutated enzymes (data not shown) indicates that the two proteins
have roughly the same secondary structure content.
NMR analysis of a protein such as AKe (23,587 Da, flexible domains, high
-helix content) is a difficult task. The proton resonance assignment was facilitated by the large spectral dispersion in the fingerprint region corresponding to
-strand residues and by the strong sequential connectivities between
C
Hi and NHi+1 in this
type of secondary structure (16). Using several two-dimensional homonuclear NMR spectra (DQF-COSY, TOCSY, and NOESY) we assigned the
majority of the resonances corresponding to the two
-sheet domains
in the AKe and AKC4. The quality of
the two-dimensional spectra and some of the sequential NOE pathways of
AKC4 are shown in Fig. 2. The
chemical shift values for the assigned resonances in the proton spectra
of AKC4 and AKe are available upon request from the authors.

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Fig. 2.
Portion of the NOESY spectrum of
AKC4 illustrating the sequential connectivities used
to assign the backbone protons in the -strands. The sample (1.5 mM) was dissolved in potassium phosphate buffer (50 mM), pH 6.5, containing 7% 2H2O
for the lock. The squares indicate the cross-peaks
corresponding to the intraresidue dipolar connectivities
C Hi/NHi and are
labeled with residue name and number. In the -strands these
interactions are weak but the position of the cross-peaks is confirmed
from comparison with correlation spectra (DQF-COSY, TOCSY). Sequential
and some of the long range connectivities are labeled with the numbers
of the two interacting residues.
|
|
Resonance assignment enabled us to identify a large number of
sequential and long range dipolar interactions between specific protons
of the protein. Observation of strong sequential connectivities d
N(i, i + 1) indicates
very short distances between the corresponding protons
(C
Hi, NHi+1), as is
the case for a backbone in an extended conformation. The local geometry
was further characterized by the analysis of the chemical shift values.
Indeed, statistical analysis of experimental NMR data on proteins,
showed that the C
protons experience a mean upfield shift of 0.4 ppm
if the residue belongs to an
-helix and almost the same downfield
shift value in the
-sheet or extended structures (17). Together, the
sequential connectivities and the secondary shift of
protons
provided a reliable delineation of the
-strands in
AKe and AKC4.
In addition, analysis of long range NOEs between resonances
corresponding to the backbone protons revealed the relative arrangement of the secondary structure elements. In particular, the presence of
long range d
(i, j)
NOE cross-peaks is characteristic for antiparallel
-sheets. The
synthesis of the present NMR data is the global topology of the
polypeptide chain in the two
-sheet domains, as represented in Fig.
3 for AKC4. A number of other long range dipolar interactions involving all the protons support this
topology. For instance, methyl protons of Leu153 give NOE
connectivities with amide protons of Tyr133,
Gly144, and Lys145 and with aromatic protons of
Tyr133.

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Fig. 3.
Schematic representation of the global
topology of the two -sheet domains in AKC4 as
defined by the present NMR analysis. The amino acids shown are
those for which at least the backbone protons were assigned. The strong
sequential connectivities (d N(i,
i + 1)) within the strands and the
d (i, j) and
dNN(i, j) long range
connectivities between C protons on opposite strands are
indicated by arrows.
|
|
The secondary and tertiary structure descriptions of AKC4,
based on the present experimental data, are very similar to that obtained for the wild-type enzyme and are completely compatible with
the crystallographic structure of AKe (4).
However, a number of minor spectroscopic modifications were observed
between the wild-type protein and AKC4 in the LID domain.
Chemical shift changes (up to 0.45 ppm) were noted for residues
adjacent to the substituted positions (Ala127,
Val125, and Val148). Also, dipolar interactions
between amide protons of Tyr133 and Thr154 and
1,2 methyl protons of Leu153 were observed
in AKC4 but not in AKe. This
suggests that Zn2+ binding in AKC4 brings
closer the two loops containing the cysteine side chains, and the
movement extends over the adjacent
-strands, so that the methyl
groups of Leu153 comes into NOE contact with the amide
proton of Tyr133. In contrast, sequential
dNN(i, i + 1)
connectivities for the amide pairs 147/148 and 148/149 and the NOE
between C
H(145) and NH(153) were observed in
AKe but not in AKC4 spectra.
Investigation of the Thermal Stability--
The heat-induced
denaturation curve in Fig. 4 was obtained
by recording the ellipticity at 222 nm, which is the minimum in the
far-UV CD spectrum for an
-helix. The two proteins show a similar
profile including an initial slow phase followed by two cooperative
transitions, separated by a clear plateau. As reflected in
the relative ellipticity changes, a comparable amount of secondary structure is unfolded in each step. The Tm (the
mid-temperature of the cooperative transition) for the first transition
of the wild-type protein is 52.5 °C which is in good agreement with
the previous data (18, 19) and close to the calorimetric value (10).
The quadruple mutant shows a significantly increased mid-temperature (61.6 °C), close to the thermal transition (63 °C) observed by microcalorimetry (10). The apoAKC4 (the four Cys mutant in
absence of Zn2+) has an intermediate thermal stability
between the wild type and AKC4 (Table
I). Dithiothreitol, an efficient
sulfhydril reductant, at 1-10 mM has no significant effect
on the denaturation transition points.

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Fig. 4.
Temperature unfolding of Ake
and AKC4 monitored by the
ellipticity at 222 nm as a function of temperature. Samples are
about 10 µM in 10 mM Tris/HCl buffer, pH 7.2. The data represent wild-type AKe
(continuous line) and the variant AKC4
(dotted line) in absence (A) and presence
(B) of Ap5A.
|
|
The second transition, not observed in previous studies, is less
cooperative and shows more similar Tm values for
the two proteins (72.6 and 75.6 °C for AKe
and AKC4, respectively). The unfolding process is entirely
reversible for AKC4 and partially reversible (80%) for the
wild-type protein when the temperature cycle is stopped after the first
transition. When the unfolding cycle is continued over this
temperature, the denaturation is irreversible.
Ap5A is a bisubstrate analog which binds simultaneously at
the ATP and AMP sites of AKs, giving a more compact structure in which
the LID domain is in close contact with the protein core (20). To
quantify the effect of its binding on the structural stability we
studied the denaturation process in the presence of an equimolar
concentration of Ap5A. The first transition is significantly shifted toward higher temperatures for
AKe, apoAKC4, and AKC4
by about 7 °C (Fig. 4 and Table I). In contrast, no effect was
observed on the second transition. A similar thermal protection was
observed for a fluorescent analog of Ap5A (19).
The temperature-induced unfolding of the wild type and two mutated
variants was further studied by recording one-dimensional proton NMR
spectra at different temperatures. The heating up to the first
equilibrium intermediate state is accompanied by continuous, small
changes of some resonances corresponding to nonexchangeable protons.
Some of these peaks were identified in the NMR one-dimensional spectra
and were assigned to precise protons of the protein (Fig. 5). One example corresponds to the
aromatic protons of Tyr181: a second peak, slightly high
field shifted and of increasing intensity is observed in all three
samples. Similarly, resonances from imidazole protons of
His172 undergo a small upfield shift while the high field
peak from Leu213 methyl (chemical shift =
0.16 ppm)
is unchanged. At the temperature where the first unfolding transition
already took place, these resonances are probes for a folded structure
as their chemical shift values are very different (from 0.36 to 0.94 ppm) from the random coil values (16). We note that Tyr181,
Leu213, and His172 are located in helices,
8,
9, and C-end of
7, respectively, within the protein core
(Fig. 1). Their chemical shift values should be largely dominated by
the ring current of neighboring aromatic side chains
(Tyr182, Tyr105, and Tyr171,
respectively). These observations reflect only small local
conformational rearrangements of the side chains within a conserved
secondary and tertiary structure. Compared with the wild type, the
spectral changes in the two variants AKC4 and
AKHC3 extend over larger temperature ranges, suggesting
that the Zn2+ binding in the LID domain influences the
local fluctuations in the central core.

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Fig. 5.
Temperature dependence of the one-dimensional
NMR spectra of three AK variants. Samples are 0.5 mM
in 50 mM phosphate buffer (pH 6.5) in
2H2O.
|
|
A large number of C
protons, resonating between 4.7 and 6.0 ppm,
conserve their low field shift characteristic for
-strand secondary
structure. An example is the C
proton of Tyr193, a
residue from the
5 strand of the parallel sheet, which is clearly
identified in all one-dimensional spectra at 5.77 ppm (Fig. 5). In the
case of AKC4, this observation is indicative of persistence
of
secondary structure in the intermediate state of the
denaturation process (at 70 °C). For AKe and
AKHC3, the native C
H are conserved during the first
thermal transition.
Coupled with the CD data, the temperature-dependent NMR
observations suggest that the parallel
-sheet and part of the
surrounding
-helices are conserved in the intermediate state and
denaturate only during the second thermal transition. Unfortunately,
due to aggregation problems, the NMR study was limited to the first part of the denaturation process.
Chemical Denaturation--
The structural stability was further
studied using urea as a chemical agent and CD spectroscopy. A single
sigmoidal transition was observed for the two studied proteins (not
shown). Curve fitting using a two-state model enabled us to calculate
the thermodynamic parameters characterizing the structural stability of
the proteins (Table I). The urea concentration at which half of the
molecules are denatured (Cm) is significantly
higher in AKC4 variant reflecting a better resistance to
urea denaturation of this protein. The calculated Gibbs free energy of
unfolding, extrapolated to zero urea concentration
(
G(H2O)) is larger in the wild type relative to the engineered variant. Thus, despite an increased thermal stability
and a higher resistance to chemical denaturation, the quadruple mutant
has a lower structural stability in standard conditions (no denaturant,
20 °C). The coefficient of linear dependence of the Gibbs free
energy of unfolding on the urea concentration (m) is
considerably decreased in AKC4, indicating a decreased cooperativity of unfolding in the engineered protein.
 |
DISCUSSION |
Structure/Function Relationships--
As indicated by the CD and
NMR results, introduction of 4 Cys residues and zinc binding in the
engineered variant AKC4 conserve the global fold of the
polypeptide chain and induce only small structural changes in the LID
domain. The Zn2+-binding loops come closer to each other
and the domain becomes more compact. The fact that the catalytic
properties are practically conserved suggests that the domain keeps its
ability to move toward the ATP-binding site. This is further supported
by the fact that Ap5A binding (which is known to favor this
movement) increases the thermal unfolding mid-temperature
(Tm) with similar extent in both wild type and
AKC4 (Fig. 4).
Mechanism of the Thermal Unfolding--
The present experimental
data provide a minimal structural basis for a basic description of the
thermal unfolding process of AKe. The CD
experiments indicate a complex thermal denaturation mechanism with two
separated cooperative steps. Comparative analysis of the NMR and CD
data suggests that the protein core, including the parallel
-sheet
and the surrounding helices (
1,
8, and
9), is conserved in the
equilibrium intermediate state and unfolds in the second step. This
domain represents about 45% of the total regular secondary structure
(
-helix and
-sheet) contributing to the CD signal around 220 nm,
in reasonable agreement with the relative extent of ellipticity change
during the second unfolding transition (Fig. 4). In agreement with this
hypothesis, Ap5A shifts significantly the
Tm of the first transition but has only a
minimal effect on the second one. Indeed, structural analysis of the
bi-substrate binding (3) revealed that the most perturbed fragments are
the connecting helices (
6 and
7) while the parallel
-sheet is
not affected. The present experiments provide no definite data on the
role of the LID domain in the denaturation process. Recent
1H/2H exchange experiments on
15N-labeled Ake2
revealed that some amide protons in the
antiparallel
-sheet exchange more rapidly than in the protein core
(parallel
-sheet,
1,
8, and
9) but more slowly than in the
peripheral
-helices. This suggests that the LID domain exhibits a
more fluctuating hydrogen bond network than the parallel
-sheet and
may unfold in early stages of denaturation. The large values of the
temperature factors observed in x-ray studies in the LID domain of the
unliganded AKe (4) support this hypothesis.
Interaction of AK with the bi-substrate Ap5A leads to a
more symmetric and compact molecule and induces a structural
stabilization of the nucleotide-closing domains (the antiparallel
-sheet, helices
2 and
3) and of the connecting helices (
6
and
7). The total accessible surface of the protein decreases by
about 13% (4) and the ligand forms tight contacts with several side
chains in the active site (20). These factors should contribute to the Ap5A-induced delay of the first denaturation step of the
two proteins described here.
Increased Thermal Stability of AKC4--
The
experiments done on apoAKC4 indicate that the four side
chain substitution and zinc binding have a cumulative effect in increasing the thermal stability. Analysis of the crystal structures suggests that the stability of the LID domain is mainly due to a
network of hydrogen bond interactions between several side chains (including the 4 substituted residues, Arg131,
Tyr133, and Glu151) keeping closer the two
small
-sheets. The 4 Cys residues (by their smaller size and polar
character) are able to integrate into this network, increasing the
global thermal stability of the protein. Zn2+ binding has
an additional contribution in restraining the global flexibility of
this solvent-exposed domain. This decreased flexibility is one of the
factors shown to differentiate the thermostable proteins from their
mesophilic counterparts (21). By an induced effect, the zinc finger
domain may also stabilize the helices connecting the domain to the
protein core (
6 and
7), preventing the fraying of peripheral
structural elements. In contrast, it has no significant effect on the
unfolding of the parallel
-sheet which takes place at higher
temperatures.
The Tm of the AK from the thermophilic B. stearothermophilus (74.5 °C) (9) is similar to that of the
second unfolding transitions reported here for
AKe and AKC4. This means that
besides the contribution of the zinc binding region there are other
contributions to the thermostability, especially related to the
external helices. Indeed, when the metal ion is removed from the
thermophilic enzyme, Tm decreases to 67 °C, a
value which is still considerably higher than in
AKe.
In contrast with the increased stability against thermal and chemical
denaturation of AKC4, its structural stability in standard physicochemical conditions appears to be lower than that of the wild-type enzyme (Table I). The explanation of this observation requires a reliable thermodynamic characterization of the protein unfolding, based on combined variation of several physicochemical factors (temperature, pH, chemical denaturant, and so forth). Also, the
complete NMR spectral assignment, the structure determination and
characterization of the internal dynamics of the proteins will provide
the necessary structural basis. These studies are currently in progress
in our laboratory.
 |
Aknowledgments |
We thank Joël Mispelter and Petya
Christova for useful discussions.
 |
FOOTNOTES |
*
This work was supported in part by grants from the Institut
National de la Santé et de la Recherche Médicale, the
Centre National de la Recherche Scientifique, Institut Pasteur, and
Institut Curie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: INSERM U350, Institut
Curie-Recherche, Centre Universitaire, Bât. 110-112, 91405 Orsay
Cedex, France. Tel.: 33 1 69 86 31 63; Fax: 33 1 69 07 53 27; E-mail:
craescu{at}curie.u-psud.fr.
1
The abbreviations used are: AK, adenylate
kinase; Ap5A,
P1,P5-di(adenosine-5')-pentaphosphate;
AKe, wild-type E. coli adenylate
kinase; AKC4, adenylate kinase variant with four amino acid
substitutions (H126C, S129C, D146C, and T149C); AKHC3,
three-substitution variant (S129C, D146C, and T149C); LID, domain
closing over ATP of AKe (residues 122-159);
DQF-COSY, double-quantum filtered correlation spectroscopy; NOE,
nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement
two-dimensional spectroscopy; TOCSY, total correlation
spectroscopy.
2
S. Burlacu-Miron and C. T. Craescu,
unpublished results.
 |
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[CrossRef]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.