From the Departamento de Biología, Facultad
de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile,
§ Instituto de Física, Universidade de São
Paulo, São Paulo, SP, Brasil, and ¶ Instituto de
Física de São Carlos, Universidade de São
Paulo, 13560-970 São Carlos, SP, Brasil
Received for publication, December 1, 2002, and in revised form, January 9, 2003
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ABSTRACT |
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The binding of MgATP and fructose-6-phosphate to
phosphofructokinase-2 from Escherichia coli induces
conformational changes that result in significant
differences in the x-ray-scattering profiles compared with the
unligated form of the enzyme. When fructose- 6-phosphate binds to the
active site of the enzyme, the pair distribution function exhibits
lower values at higher distances, indicating a more compact structure.
Upon binding of MgATP to the allosteric site of the enzyme, the
intensity at lower angles increases as a consequence of tetramer
formation, but differences along higher angles also suggest changes at
the tertiary structure level. We have used homology modeling to build
the native dimeric form of phosphofructokinase-2 and fitted the
experimental scattering curves by using rigid body movements of the
domains in the model, similar to those observed in known homologous
structures. The best fit with the experimental data of the unbound
protein was achieved with open conformations of the domains in the
model, whereas domain closure improves the agreement with the
scattering of the enzyme-fructose-6-phosphate complex. Using the
same approach, we utilized the scattering curve of the
phosphofructokinase-2-MgATP complex to model the arrangement and
conformation of dimers in the tetramer. We observed that, along with
tetramerization, binding of MgATP to the allosteric site induces domain
closure. Additionally, we used the scattering data to restore the low
resolution structure of phosphofructokinase-2 (free and bound forms) by
an ab initio procedure. Based on these findings, a proposal
is made to account for the inhibitory effect of MgATP on the enzymatic activity.
Oligomeric enzymes frequently require conformational
changes within or between subunits for their activity and regulation. Furthermore, allosteric ligands may affect enzymatic activity by means
of conformational changes to the tertiary and/or quaternary structure,
which may result in metabolic pathway regulation.
Phosphofructokinase activity, the ATP-dependent
phosphorylation of
fructose-6-P,1 is an
important step in the glycolytic pathway that is subject to strict
regulation in a wide variety of organisms. In Escherichia coli this activity is accomplished by two isozymes that differ in
their kinetic and structural properties. Phosphofructokinase-1 (Pfk-1)
has been extensively characterized and belongs to the PfkA protein
family that includes higher eukaryotic ATP-dependent phosphofructokinases ATP- and pyrophosphate-dependent
bacterial and plant phosphofructokinases (1). The atomic structure of Pfk-1 has been solved by x-ray crystallography (2); the enzyme is a
homotetramer whose state of aggregation does not change upon ligand
binding. The other isozyme, phosphofructokinase-2 (Pfk-2), presents
inhibition by the substrate MgATP when the assay is performed at low
fructose-6-P concentrations (3). Fluorescence studies demonstrated that
MgATP inhibition occurs upon binding of MgATP to an allosteric site in
Pfk-2 (4). Also, binding of MgATP promotes oligomerization of Pfk-2,
changing from a dimer to a tetramer (5-7). Such features make Pfk-2 an
excellent model to study allosteric regulation linked to protein
aggregation and enzymatic inhibition.
Pfk-2 has no conserved patterns of sequence associated with the PfkA
protein family. However, Pfk-2 is related to the PfkB superfamily of
sugar kinases which includes ribokinases, adenosine kinases,
fructokinases, and possibly, ADP-dependent glucokinases and
phosphofructokinases (8, 9). Several crystal structures have been
solved for members of this family (9-12) showing that the overall fold
is strongly conserved. For example, the root mean square deviation for
superimposed residues between E. coli ribokinase and human
adenosine kinase is 2.4 Å even though the sequence identity among them
is only 22% (11). The protein fold in this superfamily consists of two
domains, a large The comparison of the free and sugar-bound forms of ribokinase and
adenosine kinase structures reveals an important conformational change
that can be described as a hinge bending motion, with one domain
rotating toward the other by an angle of 17-30° (12, 13). The
hinge-like movement helps binding of the second substrate (MgATP) and
further catalysis. In Pfk-2, the binding of fructose-6-P promotes a
conformational change, as indicated by a 30% increment in the
fluorescence emission of the single tryptophan (Trp-88) (4) and enables
the subsequent binding of MgATP to the active site, as indicated by the
ordered bi-bi reaction mechanism of Pfk-2 in which MgATP can bind the
active site only after fructose-6-P binding (14). On the other hand,
when MgATP is bound to the allosteric site, fluorescence quenching with
a blue shift in the emission maximum is observed. It is not known
whether this last effect can be accounted for by the observed
quaternary structural transition only or if it also involves tertiary
structural changes.
To determine the effects of fructose-6-P binding to the active site and
MgATP binding to the allosteric site on the tertiary and quaternary
structure of Pfk-2, we used homology modeling combined with small angle
x-ray scattering (SAXS), a technique sensible to shape and oligomeric
state changes. In this work, SAXS data have been used to detect shape
changes upon fructose-6-P binding that have been interpreted in terms
of quasi-rigid domain movements within the dimeric model of Pfk-2.
Also, SAXS data have been used to detect changes in the aggregation
state of the enzyme upon MgATP binding, and a model for the subunit
arrangement in the tetramer is suggested. Based on our results, a
mechanism for the inhibitory effect of MgATP is proposed.
Protein Purification--
pET 21-d plasmid (Novagen) containing
the cloned original gene (15) was transformed into E. coli
strain JM109, and 2 liters of culture were grown in Luria Broth medium
supplemented with ampicillin to a final concentration of 100 µg/ml.
Protein expression was induced at A600 = 0.6 by
the addition of isopropyl-1-thio- Homology Modeling--
Homology models of Pfk-2 were constructed
with the program MODELLER (Version 6) (17), which implements an
automated approach to comparative protein structure modeling by
satisfaction of spatial restraints. To find template structures, a
position-specific matrix was iteratively generated searching over a
non-redundant protein sequence data base with the PSI-Blast program
(18) using BLOSUM62 as the starting matrix and an expectation value
threshold of 0.001 for inclusion in the next iteration. The convergence
matrix was used to search the Protein Data Bank (PDB) amino acid
sequence data base producing significant alignments with E. coli ribokinase (PDB code 1RK2; similarity 37%, expectation value
3 × 10 Solution Scattering Experiments and Data Processing--
SAXS
data were collected at the SAS beam line of the National Synchrotron
Light Laboratory (Campinas, Brazil) using a one-dimensional position-sensitive detector (22). Experiments in the absence of ligands
and in the presence of MgATP were done at 2, 4.2, and 30 mg/ml of
Pfk-2; experiments in the presence of fructose-6-P were performed at 5 and 25 mg/ml of Pfk-2. A wavelength
The scattering curves of the protein solutions and the corresponding
buffer were collected in several frames (15 s to 3 min) to monitor
radiation damage and beam stability. The data were normalized to the
intensity of the incident beam and corrected for non-homogeneous
detector response. De-smearing for the 8-mm height detector entrance
window was also performed. The scattering of the buffer was subtracted,
and the resulting curves were scaled to equivalent concentrations. To
provide curves free from concentration artifacts, we used samples with
protein concentrations of 2-5 mg/ml to build the low resolution part
of the SAXS curve (q < 0.15 Å Ab Initio Shape Determination--
The resolution of the
resulting solution x-ray-scattering curve extended to 11.4 Å. The low
resolution protein shape was restored using the ab initio
procedure described by Svergun (25) as implemented in the program
GASBOR. In this method, a dummy residue model is generated by a
random-walk C Domain Movement Modeling--
The open and closed
crystallographic structures of ribokinase (PDB codes 1RKA and 1RKD,
respectively) were chosen as templates to model the domain movements in
Pfk-2. Because these PDB entries contain coordinates for just one
monomer of ribokinase, the second monomeric subunit of the dimeric
ribokinase was obtained by application of the appropriate 2-fold
crystallographic symmetry operations. The program DynDom (26) was then
used to define the quasi-rigid domains, the flexible inter-domain
connecting residues, and the screw axes that virtually describe the
conformation transition in ribokinase. Using a sliding window of 5 residues, the program DynDom found three dynamic domains, corresponding to the two Tetramer Modeling--
Two copies of each dimeric model (see
above) were aligned putting their centers of mass (COMs) at the origin
of an orthogonal reference system, with their 2-fold symmetry axes
aligned along x and their major inertia axis aligned along
y (program MOLEMAN2). After rotation of one of the dimers by
180° along y, the dimers were translated in opposite
directions along x, obtaining the two major configurations
of the tetramer (tetramer-I and tetramer-II) depending on the direction
of translation. Several tetrameric models were obtained applying
different separations (along x) between the COMs of the
dimers (31-39 Å for tetramer I, 26-34 Å for tetramer II, 0.333-Å
translation step). Furthermore, several shear angles (rotations in
opposite directions along x) were applied to each dimer
(0-44° with 4° step). For each configuration, the fit against SAXS
data from Pfk-2 in complex with MgATP was evaluated as described below.
Calculation of SAXS Intensity from Atomic Models and Estimation
of the Uncertainty of the Fitted Parameters--
The theoretical SAXS
curves were determined from the atomic models using the program CRYSOL
(27). The program calculates the SAXS intensity through the
equation,
Two parameters, the excluded volume of the particle (V) and
the electron density in the hydration layer (
To estimate the errors in the parameters fitted during dimer and
tetramer modeling (i.e. the Homology Modeling
The three-dimensional structure of dimeric Pfk-2 was constructed
by homology modeling (program MODELLER-6) (17) using the crystal
structure of E. coli ribokinase as template (37%
similarity), as described under "Experimental Procedures." Ten
models of Pfk-2 were built starting from different random initial
atomic positions at the beginning of the optimization and evaluated by
pseudo-energy parameters. The C
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
domain and a small domain. In ribokinase this
small domain is a
-sheet that acts as a lid over the active site and
also as the dimerization interface. Adenosine kinases have
-helical
insertions in the small domain so that dimerization is precluded.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside to
a final concentration of 0.5 mM. After 4 h of
induction, cells were collected by centrifugation. Purification was as
described (16) using the above cell pellets as the starting point.
60), human adenosine kinase (PDB code 1BX4;
similarity 29%, expectation value 2 × 10
51), and
Toxoplasma gondii adenosine kinase (PDB code 1DH0;
similarity 29%, expectation value 5 × 10
34). This
alignment was further corrected using information derived from a
structural alignment of these structures. The final alignment was used
as input for MODELLER with 1RK2 as template, since it is the only
dimeric homologue. Ten models were built using the MODELLER default
routine in which initial coordinates are randomized in Cartesian space
by 4 Å before variable target function optimization and evaluated by
pseudo-energy parameters. The probability of a good model structure,
p(G), associated to the ratio between PROSA II
Z-score (19) and the natural logarithm of its sequence length (20), was calculated on the pG server (guitar.rockefeller.edu/pg). The three-dimensional-one-dimensional score of the
models was calculated using the Verify3D program (21).
= 1.488 Å was used, with
sample-detector distances of 631.2 and 1260 mm providing two scattering
curves that, combined, covered the momentum transfer range 0.01 Å
1 < q < 0.55 Å
1
(q = 4
sin
/
, where
is half the scattering angle).
1) and
25-30 mg/ml for the high resolution part (0.15 Å
1 < q < 0.55 Å
1) of the curve shown in Fig.
2. The pair distribution functions p(r) were
evaluated by indirect Fourier transformation using the program GNOM
(23). The SAXS intensity produced by an isotropic, monodisperse dilute
solution of proteins, in the limit for small q, obeys the
Guinier approximation (24),
where I (0) is proportional to the molecular weight
provided I(q) is normalized to an equivalent
protein concentration, and Rg is the protein radius
of gyration, defined by,
(Eq. 1)
where Zk is the atomic number of the atom
located at a distance rk from the electronic center
of mass. The radii of gyration of the Pfk-2 molecules in their
different states were calculated from the slopes of the Guinier plots
(ln I(q) versus
q2).
(Eq. 2)
chain and is folded in a way to minimize the
discrepancy between the calculated scattering curve from the model and
the experimental data. The program simulates the protein internal
structure, which makes it unnecessary to subtract a constant from the
experimental data to ensure Porod's law (24). Several runs of ab
initio shape determination with different starting conditions led
to consistent results as judged by the structural similarity of the
output models, yielding nearly identical scattering patterns and
fitting statistics in a stable and self-consistent process. The final
shape restoration for the Pfk-2 free protein and with fructose-6-P was
performed using 618 dummy residues and 611 waters assuming 2-fold
molecular symmetry. In the case of Pfk-2 with an excess of MgATP we
used 1236 dummy residues and 988 waters assuming a 222 molecular symmetry.
/
domains of the dimer (residues 4-13, 42-96, and 118-309 from each monomer, following the 1RKD numbering scheme) and
the central
barrel to which both subunits contribute (residues 18-38, 99-113). Residues 14-17, 39-41, 97-98, and 114-117 in each monomer were defined as composing a flexible hinge that rotates one
/
domain toward the central
barrel. The directions of the two
screw axes (one for each monomer) have been found as well as the
associated rotation (~17°) and translation (~1 Å) parameters. Finally, the Pfk-2 model was superimposed on the ribokinase structure (using just the homologous residues in the central
barrel domain as
reference), and the residues corresponding to the two
/
domains were rigidly rotated along the screw axes found by DynDom for ribokinase. Several models were generated (program MOLEMAN2;
x-ray.bmc.uu.se/usf/moleman2_man.html) rotating the
/
domains
from
20° (most closed model) to +40° (most open model) with an
angular step of 2°. The theoretical SAXS curves of each rotated model
were subsequently fitted to the experimental data as described under
"Calculation of SAXS Intensity from Atomic Models."
where Aa(q) is the amplitude
scattered by the protein (calculated from the atomic structure
factors),
(Eq. 3)
sAs(q) is the
amplitude produced by the excluded volume (determined using dummy
Gaussian spheres placed at the atomic positions), and
s is
the electron density of the solvent. The first solvation shell is
modeled by a hydration shell that yields an amplitude
bAb(q), where
b is the electron density difference between the
hydration shell and the solvent. The hydration shell is approximated by
a 3-Å-thick uniform layer placed 2 Å away from the protein envelope.
The symbol < >
indicates spherical averaging.
b), are optimized by the program CRYSOL to minimize the discrepancy
defined
as,
where Iexp(qj) and
(Eq. 4)
(qj) denote the experimental SAXS intensity of
the jth point and its standard deviation, respectively, and
N is the number of experimental points. The excluded volume
is varied around the value predicted from the molecular mass by
changing the average displaced volume per dummy atom to account for the
uncertainty in its partial specific volume.
/
domain rotation angle,
the dimer shear angle, and the dimer COM distance), the
F-statistics method was used (28). The
F-statistics parameter, as applied to the present work, can
be written as,
where N is the number of experimental points used by
the fitting procedure (n = 89 in this work),
nc = 2 is the number of the
CRYSOL fitted parameters (i.e. V and
(Eq. 5)
b), and nm is the number of parameters
varied during modeling (nm = 1 for dimer modeling
and nm = 3 for tetramer modeling).
F
, as defined in Equation 5, is a
measure of the relative improvement of
when the simple CRYSOL
fitting procedure with a single "fixed" molecular structure is
substituted by the evaluation of several "variable" structures. A
given increase in F
(reduction in
)
due to the introduction of nm additional parameters
is statistically more than 95% significant if
F
> 9.55 (for nm = 1) or
F
> 5.79 (for nm = 3)
(28). The parameter errors reported in this work are relative to the
95% confidence limit of the F-statistics.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain trace of the 10 models of
Pfk-2 are superposed in Fig. 1a, showing
the positional variability between the models (only the monomer is
shown for simplicity). The probability of good global folding,
p(G) (20), was around 0.999 for each model, and
the average three-dimensional-one-dimensional profile score (program
Verify3D) (21) was >0.1 for 94% of all sequence positions using a
21-residue sliding window (Fig. 1b).
View larger version (30K):
[in a new window]
Fig. 1.
Homology modeling of Pfk-2.
a, superposition of the C -traces of 10 models of
monomeric Pfk-2 generated by MODELLER. b, average Verify3D
score profile for the models shown in a. The dotted
line indicates a score of 0.1. The molecular models were drawn
using the program PyMOL (Delano Scientific, San Carlos, CA,
www.pymol.org).
The Pfk-2 model presents the two-domain structure described for
ribokinase (10). The larger domain forms an /
/
three-layer sandwich, which we call the "
/
domain," whereas the smaller domain forms a
-structure responsible for interfacial contacts in
the dimer. The active site lies in the cleft formed by both domains,
and the single tryptophan, Trp-88, is located in the region between
both domains. The lowest three-dimensional-one-dimensional score region
in the profile is located around position 120 (Fig. 1b).
This region also has a low score in the experimental ribokinase structure (not shown) and corresponds to a distorted
-helix. The
score worsens slightly in the Pfk-2 models, where there is a one-amino
acid insertion in the primary structure.
Solution Small Angle X-ray Scattering Curves
Three sets of SAXS measurements were carried out with Pfk-2,
without ligands, and in the presence of saturating concentrations of
fructose-6-P or MgATP. All experimental curves are plotted in Fig.
2. The existence of induced
conformational changes in the Pfk-2 structure is clearly observed when
fructose-6-P or MgATP is bound to the enzyme. Differences in SAXS
intensities can be observed along the whole q range. In the
case of the Pfk-2-fructose-6-P complex differences are small but not
negligible.
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Differences in the scattering curve for q < 0.08 Å1 upon MgATP binding indicate a change in the
oligomeric state of the Pfk-2 with a 2-fold increment in scattering
intensity at q = 0, I(0), relative to the free and fructose-6-P-bound forms. When no ligands are
bound to Pfk-2, its radius of gyration is 29.10 ± 0.21 Å. Upon
MgATP binding to the allosteric site, the radius increases up to
35.38 ± 0.14 Å as a consequence of tetramer formation. The same
difference in radius of gyration upon MgATP binding was observed previously in dynamic light-scattering experiments (7). On the other
hand, when fructose-6-P is bound, the protein radius of gyration
shrinks slightly to 28.67 ± 0.19 Å.
Previous experimental results (5-7) indicate that binding of MgATP to
the enzyme promotes a change in quaternary structure. The deep valley
observed in the scattering intensity curve in the presence of MgATP,
around q = 0.2 Å1 (Fig. 2), suggests
that the ligand also affects Pfk-2 at the tertiary structure level.
This feature is not observed in the SAXS curve of the unbound enzyme.
When fructose-6-P binds to Pfk-2, a conformational change associated to
the tertiary structure of the protein occurs. This change is apparent
in the pair distribution function of the protein, p(r), determined from the SAXS results, which is
plotted in Fig. 3. This function exhibits
lower values at distances between 30 and 50 Å in Pfk-2 complexed to
fructose-6-P relative to the free form. It is therefore expected that
Pfk-2 should be somewhat more compact when the sugar is bound to the
active site. To obtain more detailed information about these changes,
we have performed rigid body refinements of the domain orientations in
the model, as described below.
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Modeling Ligand-induced Conformational Changes Using SAXS Data
Free and Fructose-6-P-bound Forms of Pfk-2--
The SAXS data
corresponding to the free and the fructose-6-P-bound forms of Pfk-2
indicate a probable substrate-induced conformational change of the
enzyme structure. Substrate-induced conformational changes based on
crystallographic results have already been reported for the related
enzymes ribokinase and adenosine kinase (12, 13). These enzymes exhibit
a hinge-like movement of the /
domain(s) toward the small domain
(lid). This movement can be described as a quasi-rigid rotation around
an axis passing through the connecting interdomain residues, although
the rotation direction and angle are somewhat different for the two
enzymes. To account for the different experimental SAXS curves obtained
from Pfk-2 in the absence and in the presence of fructose-6-P, our
Pfk-2 three-dimensional structure model has been modified by several rigid rotations of each of the two
/
domains around an axis equivalent to the domain rotation axis found in ribokinase, which is
evolutionary more closely related to Pfk-2 than adenosine kinase.
Fig. 4b shows the -fit to
the SAXS data of the calculated scattering intensity for the proposed
models at different
/
domain rotation angles. In this figure, the
rotation angles 0° and +17° correspond to the closed and open
conformations of the original ribokinase structure, respectively. Also,
a rotation of 0° corresponds to the original Pfk-2 homology model. It
can be seen that, for the unbound form of Pfk-2, the lowest discrepancy
is obtained using a more open conformation, with a clear minimum angle
around 25° (± 7°). For the fructose-6-P-bound form, a better fit
is obtained using a closed conformation with a minimum around 13°,
although in this case the minimum of the
function is less well
defined, corresponding to a rather large uncertainty (±12°). This
suggests that Pfk-2, upon fructose-6-P binding, adopts a more closed
conformation than the free form, similarly to that observed for
ribokinase with ribose. The rotation angles corresponding to the
minima in Fig. 4b do not correspond exactly to the free and
substrate-bound structures of ribokinase. This may reflect an actual
different opening angle of the structure in the free and
substrate-bound form. This is not totally unrealistic; a comparison of
the substrate-bound forms of adenosine kinase and ribokinase reveals
that the former is more open by a rotation angle of ~6 degrees,
indicating that the closed conformations of this family of proteins may
have different orientations of the
/
domains. Alternatively, the
enzyme may present movements that are different from those proposed in
our model.
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MgATP-bound Form; Tetrameric Packing of Pfk-2--
It is known
that MgATP binding induces tetramerization in Pfk-2, but the quaternary
structure of the tetrameric enzyme in solution remains unknown. Using
our SAXS data, we have modeled the tetrameric arrangement of the enzyme
as being a "dimer of dimers." This implies a 222 point symmetry for
the tetramer (D2, according to Schoenflies
notation), where one of the 2-fold axes corresponds to the symmetry
axis of both dimers (the x axis in Fig.
5). This axis passes through the two
central -barrels.
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Two opposite orientations of the dimers are possible (Fig. 5); one
(which we called tetramer-I) with the active sites looking outward from
the tetramer and the other (tetramer-II) with the active sites looking
inward. Fig. 6b shows the
-fit to the experimental SAXS data for the tetrameric models of type
I and II. The tetrameric models have been obtained using the dimeric
models previously described in this article, i.e. using
different opening angles of the monomers (rotation of the
/
domains, see Figs. 4a and 5). The
-fit in Fig.
6b refers to optimized tetrameric models. The distances
between the COMs of the dimers and the shear angle (Fig. 5) of the
dimers along their symmetry axis have been refined to get the best fit
to the SAXS data (Fig. 6a). These are the only rigid
movements of the dimers that are allowed in order to maintain the 222 symmetry of the tetramer.
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The discrepancy from the experimental SAXS data obtained for the best
fitted model is small (
1) both for tetramer-I and for
tetramer-II. It is evident that, no matter which orientation (tetramer-I or -II) is used, the best fits with SAXS data are achieved
with closed conformations of the monomers, similar to what is observed
for the complex with fructose-6-P. Although the
curve for
tetramer-II shows lower values than that for tetramer-I, inspection of
the three-dimensional models indicates that these lower
values are
reached at the expense of unrealistic clashes between the two dimers in
tetramer-II (not shown). Furthermore, tetrameric models of type II,
built with a reasonable distance between the dimers to prevent steric
hindrance, poorly fit the SAXS data (
> 3). On the other hand,
the minimum
for tetramer-I reflects a configuration that could
reasonably represent (within the limits of our rigid-body modeling of
the domains configuration) the real structure.
Fig. 6a shows the -fit as a function of the COM distance
and the shear angle between the dimers in tetramer-I (using a domain opening of 7°). Considering the three variables of our rigid-body modeling for the tetramer (COM distance between the dimers, shear angle
of the dimers, monomer aperture), the region around the minimum
appears to be relatively flat. As a matter of fact, the 95% confidence
limits, according to the F-statistics, correspond to COM
distances between 34.7 and 36.3 Å, shear angles between 4 and 16°,
and monomer opening angles between 1 and 9° around the minimum
(maximum
variation from 1.19 to 1.23). The parameter that mostly
affects
is the distance between the dimers. The opening and the
shear angle, within the ranges mentioned above, do not significantly
modify the
value. However, the discrepancy increases quickly
outside these ranges.
Inspection of the proposed tetrameric model indicates that the
dimer-dimer interface should be formed by contacts between the /
domains from opposite monomers, although the fine details of this
interaction cannot be deduced from our simple, low resolution model.
Ab Initio Low Resolution Structures
The SAXS data from the free, fructose-6-P, and MgATP-bound forms
of Pfk-2 were used to restore the low resolution shape of the protein
using the dummy residue model method of Svergun et al. (25),
as described under "Experimental Procedures." The restored shape
for each conformation of Pfk-2 yields a good fit to the experimental
data (see the legend of Fig. 2), indicating that imposed symmetry
restrictions actually reflect prevalent features in the structure. The
molecular envelope of each ab initio model is shown
superimposed to the corresponding refined homology model in Fig.
7, showing good agreement between both
kinds of structures.
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DISCUSSION |
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Domain closure induced by sugar binding has been demonstrated in crystallographic high resolution studies on ribokinase and adenosine kinase (12, 13). These domain motions are characterized by large changes in main chain torsion angles of a small number of residues that comprise the hinge that separates the small and large domains of these enzymes. In adenosine kinase, comparison of dihedral angles of the free and sugar-bound structures reveals that two glycines (Gly-68 and Gly-69) undergo the large torsional changes necessary for hinge bending. When sugar binds to its site, displacement of these glycines occurs to avoid steric hindrance. The nearly absolute conservation of these glycine residues throughout the PfkB superfamily indicates that they probably play the same critical role in hinge bending. In addition, sugar binding affects the nearby nucleotide binding site in ribokinase, shifting it toward a conformation that is observed when nucleotide is bound, possibly increasing the affinity for this substrate. The main feature of domain closure is the complete occlusion of the sugar site from bulk solvent.
These observations suggest a general mechanism for the reaction
catalyzed by kinases in this superfamily; sugar binds initially in an
open active site, favoring domain closure, affecting the nucleotide
binding site, and therefore, increasing its affinity for ATP. Three
independent lines of evidence corroborate these predictions about
structural changes of Pfk-2 upon sugar binding. First,
previous kinetic studies show that a compulsory ordered kinetic
mechanism occurs in Pfk-2, in which MgATP binds the active site only
after fructose-6-P binding (14); therefore, sugar binding increases the
affinity for MgATP in the active site. Second, and supporting domain
closure, the structure of Pfk-2 in solution appears to be more compact
when it is bound to fructose-6-P, as suggested by limited proteolysis
experiments (7), where binding of fructose-6-P increases the resistance
to cleavage by several proteases. And third, rigid body /
domain
closure of the homology model improves the agreement with the
experimental SAXS data of the fructose-6-P-bound form.
Based on the present results, we also propose a model for the effect of MgATP on the enzymatic activity of Pfk-2. As can be seen from Fig. 5b, a closed structure is locked when MgATP is bound to the allosteric site no matter what configuration (tetramer-I or -II) is used to fit experimental data. By analogy with the ribokinase and adenosine kinase closed forms, it might be expected that occlusion of the sugar site from solvent hinders either fructose-6-P binding or product release, thus giving the first structural support to a mechanism for MgATP enzymatic inhibition in this enzyme.
It should be noticed that if domain closure exposes surface
determinants needed for tetramerization, oligomerization would be
expected also as a consequence of fructose-6-P binding, but this is not
the case. Unless MgATP itself takes part in the interaction surface,
this ligand must induce another conformational change different from
the /
domain closure to induce tetramerization. In this regard,
the calculated scattering curves from tetrameric models (not shown)
does not fit very well the valley around q = 0.2 Å
1 in the experimental data from the Pfk-2-MgATP complex
(Fig. 2). In ribokinase, the dimer interface bears striking similarity
to domains of two ligand binding and transport proteins that are built
up from orthogonal
sheets (29), but ribokinase lacks an internal
space large enough to hold a small molecule ligand. Unfortunately, the
existence of such appropriate internal space for MgATP binding in the
Pfk-2 dimer interface cannot be established by the present work.
Chemical modification studies suggest that cysteine 295 is involved in the dimer-dimer interface.2 Mapping this amino acid onto the three-dimensional structure of the proposed tetramer-I model indicates that, although this residue is not located at the interface, it occupies a nearby location.
In previous fluorescence studies of the single tryptophan in Pfk-2
(Trp-88), three states have been reported (4), one with a high quantum
yield (Pfk-2 saturated with fructose-6-P), another with an intermediate
emission (Pfk-2 without ligands), and a third one with a low quantum
yield state (Pfk-2 in presence of MgATP). Acrylamide quenching of
protein fluorescence demonstrates that the tryptophan solvent
accessibility is reduced when Pfk-2 is bound to MgATP as compared with
the free enzyme and the Pfk-2-fructose-6-P complex, whose
accessibilities are similar. Our SAXS results fully agree with these
previous observations; in the model proposed here, Trp-88 is located in
the /
domain near the hinge; thereby this intrinsic probe could
directly detect different openness of domains in monomers. In the
tetramer model, Trp-88 is located near the dimer-dimer interface; thus,
solvent accessibility might be reduced as a consequence of tetramerization.
Our model refinement of dimeric Pfk-2 in its free and fructose-6-P-bound states using SAXS results indicates that the sugar promotes a domain closure with ~12 degrees of rotation. Results of our modeling indicate that the tetrameric structure of Pfk-2 complexed with MgATP is composed by two parallel or slightly misaligned dimers located at a distance of around 34 Å between each COM, with the active sites looking outward from the tetramer and the monomeric subunits in an almost closed conformation. This tetrameric model provides satisfactory agreement with previous studies on intrinsic fluorescence and chemical modification of the enzyme. It represents the result of a combination of theoretical homology modeling and experimental low resolution structure determination, which we consider to be valuable in the absence of a crystal structure.
Most hinge-bending proteins appear to display a dynamic equilibrium
between their open and closed states, the latter stabilized by ligand
binding. Because the present SAXS measurements of E. coli
Pfk-2 were taken under equilibrium conditions, our observations could
be interpreted as follows. In solution, with no ligands added,
equilibrium is displaced toward an open conformation. When fructose-6-P
binds the active site, closed structures become populated, helping the
subsequent binding of MgATP to the active site, the first step toward
catalysis. However, binding of MgATP to the allosteric site promotes,
along with tetramerization, a domain closure that occludes the active
site, and as a consequence, this impedes the entrance of fructose-6-P
to the active site (or the release of products), thus producing the
observed enzymatic inhibition.
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ACKNOWLEDGEMENTS |
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We acknowledge Mauricio Baez and Dmitri Svergun for useful discussions.
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FOOTNOTES |
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* This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico, Chile Grant 1010645 and Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil Grants 99/09471-7, 01/07798-0, and 98/14138-2. We also thank Programa de Apoio a Núcleos de Excelência (Ministerio de Ciência e Tecnologia, Brazil), Brazil and National Synchrotron Light Laboratory (Campinas, Brazil) for financial support.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. Tel.:
56-2-6787450; Fax: 56-2-2726006; E-mail: jbabul@uchile.cl.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M212137200
2 M. Baez, V. Guixé, and J. Babul, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: fructose-6-P, fructose-6-phosphate; Pfk, phosphofructokinase; SAXS, small-angle x-ray scattering; COM, center of mass.
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