 |
INTRODUCTION |
Tubulins are GTP-binding proteins that play central roles in
eukaryotic cell division and organization. The 
-tubulin dimers reversibly assemble to form the microtubules. The closest relatives of
tubulins are the predicted homologous bacterial cell division FtsZ
proteins (1). The GTP bound to the
-subunit is exchangeable in the
dimer (E-site1; Ref. 2), and
is hydrolyzed to GDP and Pi as a result of microtubule formation. The nucleotide
-phosphate and a coordinated
Mg2+ ion control the assembly activity of tubulin and
microtubule stability (3-6). Tubulin with GDP in the
-subunit
(GDP-tubulin) is unable to assemble into microtubules except by ligand
binding to the paclitaxel site (7). GDP-tubulin is in an inactive
conformation (8, 9) which favors curved assembly into double rings
corresponding to pairs of curved protofilament segments (10, 11), and
the curling of exposed protofilaments at microtubule ends (12, 13). In
contrast to
-tubulin, the molecule of GTP bound to the
-subunit is considered non-exchangeable (N-site; Ref. 2), stays essentially bound during the entire life of the protein suggesting that it may be a
structural cofactor of tubulin (14), and is coordinated to a slowly
dissociating divalent cation (4).
Magnesium ions have a well established influence on tubulin-nucleotide
interactions (3, 4, 15, 16) and on tubulin self-association (17, 18),
including microtubule assembly (19). Equilibration in
Mg2+-free buffers results in a partial release of the GTP
bound, followed by an irreversible loss of activity (4, 20). Previous
studies of divalent cation binding (3, 4, 17, 21, 22) indicated that
tubulin has two classes of Mg2+ binding sites, one of high
affinity (with an association binding constant,
K1,Mg, in the order of 106
M
1) and the other of low affinity
(K2,Mg, 102 to 103
M
1). The stoichiometry of the first class of
sites depends on the nucleotide bound to the E-site; GTP-tubulin has
two tightly bound Mg2+ (at the N- and E-sites), whereas
GDP-tubulin has a single high affinity Mg2+ (N-site; the
E-site becomes low affinity (see Ref. 3)). This has been confirmed by
studying the binding of Mg2+ to tubulin having GTP, GDP, or
no nucleotide at the exchangeable site of the
-subunit and one
Mg2+ ion already bound (23). The low affinity
Mg2+ binding sites are involved in tubulin polymerization
(19) and in the equilibrium association of the 
-dimer (8). In
contrast, neither the high affinity binding of Mg2+ to the
N-site nor its intriguing role are well understood (3, 4, 23, 24).
The present study aims to understand the specific roles of the
respective Mg2+ ions coordinated with the GTP bound to E-
and N-sites in tubulin stability, structure and function. Toward these
purposes, the isotherm of binding of Mg2+ to the N-site has
been measured, and the different effects of the high affinity cations
bound to GDP- and GTP-tubulin have been compared employing DSC, CD,
fluorescence, and sedimentation equilibrium methods. It will be shown
that the functional microtubule-stabilizing cation and
-phosphate at
the E-site impart negligible stabilization to the 
-tubulin dimer,
whereas the non-functional cation bound to the N-site, at the
-subunit, is essential for tubulin stability, and communicates with
the colchicine site at the
-subunit.
 |
EXPERIMENTAL PROCEDURES |
Preparation of calf brain tubulin, without (GDP-tubulin) or with
(GTP-tubulin) a
-phosphate at the E-site was performed as described
in Ref. 7, with minor modifications. GDP-tubulin was finally
equilibrated in PEDTA buffer with 1 mM GDP by
chromatography in Sephadex G-25 columns (10 or 25 × 0.9 cm). To
prepare GTP-tubulin, 1 mM GTP and Mg2+ were
added to GDP-tubulin. In the experiments at the lower free Mg2+ concentration, the EDTA concentration in the buffer
was 2 mM. Nucleotides and Mg2+ quantification
by high performance liquid chromatography and atomic absorption
spectrometry, respectively, and microtubule assembly were performed as
described (7, 25), unless otherwise indicated. Fresh MilliQ grade water
was employed to prepare all the solutions, as well as plasticware
containers; any glassware material was rinsed with PEDTA buffer before
use.
Binding of Mg2+ to Tubulin--
The binding of
Mg2+ was measured as follows. Aliquots of 200 µl of
tubulin (15 µM) with a known total Mg2+
concentration were incubated at 10 °C for 30 min and centrifuged at
100,000 rpm for 1 h in a TLA-100 rotor, using a TLX-120
ultracentrifuge (Beckman Instruments Inc.). After centrifugation, the
lower half of the tubes, which contain tubulin in equilibrium with free
Mg2+, and the upper half, with cation and essentially no
protein, were carefully withdrawn, and the total Mg2+
concentration was determined in both halves. Mg2+ bound to
tubulin was quantified by the difference in the cation concentration
between the lower and the upper parts of the centrifuge tube. Free
Mg2+ was calculated from the total Mg2+
concentration in the upper half, by solving the multiple equilibria, which take into account the cation binding to phosphate, nucleotide, and EDTA. The stability constants for Mg2+ complexes at pH
7.0 employed were as follows: phosphate, 68 M
1 (26, 27); GDP, 607 M
1 (3); GTP, 2830 M
1 (3); and EDTA, 2.5 × 105
M
1 (28). These values are within 5%
variation with respect to other constants reported in the literature
(26, 29-32). This, together with the calculated errors in the
measurement of the total amount of Mg2+, resulted in an
estimated uncertainty of 10-15% for the lower calculated free
Mg2+ concentrations, and less than 10% for the higher
concentrations. Note that, in the buffer solution employed, the small
free Mg2+ concentration approximates the mean ionic
activity of MgCl2 within experimental error.
A model of ligand binding assuming multiple classes of independent
binding sites for Mg2+ (33) in the tubulin molecule was
fitted to the experimental data, using a non-linear least squares
procedure, based on the modified Nelder-Mead simplex algorithm
(34).
Time Course of Tubulin Inactivation--
The effect of
Mg2+ on the kinetics of tubulin inactivation at constant
temperature was followed by monitoring two independent properties: (i) the loss of the assembly capacity of tubulin (20 µM) with paclitaxel, monitored turbidimetrically (7) and
(ii) the loss of colchicine binding sites, measured from the
fluorescence of the MTC-tubulin complex (see below). Before
measurement, samples were supplemented to final total Mg2+
concentrations of 7 and 5 mM in the assembly and MTC
binding buffers, respectively. Control experiments were run in parallel at 7 mM Mg2+ in the initial equilibration
buffer.
Circular Dichroism--
The far-UV CD spectra of tubulin (1-5
µM, equilibrated in PEDTA with 1 mM
nucleotide and a known amount of Mg2+) were acquired in a
JASCO J720 dichrograph equipped with a temperature regulated cell
holder (1, 35), with a 0.1-cm cell at 20 ± 1 °C. Thermal
denaturation was monitored following the variation in ellipticity at
220 nm, using a temperature scan rate of
0.5 °C·min
1. Changes in secondary structure were
estimated by deconvolution of the CD spectra using Yang (36), LIMCOMB,
and CCA (37, 38) methods.
Fluorescence of the MTC-Tubulin Complex--
The effect of
Mg2+ on the fluorescence spectrum of the colchicine analog
MTC (50 µM total concentration) bound to tubulin (5 µM) was measured essentially as described (39), using a
Shimadzu RF-540 spectrofluorimeter (Kyoto, Japan;
ex = 350 nm,
em = 423 nm). The fluorescence cell (5 × 10 mm) was mounted on a holder thermostated with a water bath at
20 °C. The free and bound ligand were measured with the same
high speed centrifugation method described for Mg2+
binding, except that MTC was measured spectrophotometrically (
343 = (1.76 ± 0.01) × 104
M
1 cm
1) (39).
Analytical Ultracentrifugation--
The measurements were
performed at 10 °C with a Beckman Optima XL-A analytical
ultracentrifuge equipped with absorbance optics, using an An60Ti rotor
and either 12-mm double sector or six-channel centerpieces. Tubulin
samples (loading concentrations between 0.5 and 15 µM)
were equilibrated in PEDTA, 20 µM nucleotide, with the
desired amount of Mg2+. Short column (40-50 µl)
sedimentation equilibrium was performed either at low speed (30 min at
30,000 rpm, followed by 2-3 h at 15,000 rpm) or as described (40):
1 h at 32,000 rpm, followed by 1-2 h at 26,000 rpm, which
permitted attainment of equilibrium. Absorbance scans were taken at the
appropriate wavelength (230, 275, or 290 nm). In all cases, base-line
offsets were determined subsequently by high speed sedimentation.
Whole-cell apparent weight-average molecular masses
(Mw,ac) were obtained using the
programs XLAEQ and EQASSOC (supplied by Beckman; see Ref. 41). The
partial specific volume was 0.736 cm3/g (42), which was
corrected for temperature (43).
To determine the equilibrium constant for tubulin dimerization
(K2), two different methodologies were employed.
(i) Equilibrium association models were globally fitted to multiple
sedimentation equilibrium data using either the MicroCal-Origin version
of NONLIN (44) or the programs MULTEQ1B and MULTEQ3B based on the
conservation of signal algorithm (41). A value of 1.16 ml
mg
1 cm
1 was used for the extinction
coefficient of tubulin at 275 nm in phosphate buffer (45), and the
subunit relative molecular mass was taken as 55,000 (46). (ii) The
dependence of the apparent weight-average molecular mass (Mw,a)
on protein concentration was calculated from the local slopes of
transformed data (lnC versus r2) at defined
radial distance intervals, using the program MWPLOTZ (kindly supplied
by A. Minton, National Institutes of Health, Bethesda, MD). In this
study, the Mw,a values were calculated by superimposing data
obtained from different loading protein concentrations and averaging
over a concentration interval of ±0.1 log units. Models for
self-association (47, 48) were fitted to the Mw,a
versus concentration data using a non-linear least-squares method (34).
Sedimentation velocity experiments were performed at 42,000 and 60,000 rpm. Sedimentation coefficients were calculated from the rate of the
movement of (i) the solute boundary (with XLAVEL, Beckman) or (ii) the
second moment of the boundary (with VELGAMMA, Beckman), and (iii) from
the distribution of the apparent sedimentation coefficients,
g(s*), using the DCDT program (49, 50). The sedimentation coefficients were corrected to standard conditions (51)
to get the corresponding s20,w
values.
Differential Scanning Calorimetry--
The heat capacity
measurements were performed in a MicroCal MC2 differential scanning
calorimeter, as described previously (52). Tubulin samples (15 µM) for DSC were equilibrated in PEDTA buffer with 1 mM GDP (or GTP) and the desired Mg2+
concentration. The scanning rate was 0.5 °C min
1,
unless otherwise stated. The reversibility of thermal transitions was
checked by reheating the samples after the first scan. The influence of
scanning conditions on the profiles of the calorimetric transitions of
tubulin was checked by running samples at several rates. The kinetic
analysis of the DSC curves was carried out as described (53, 54).
 |
RESULTS AND DISCUSSION |
The thermal stability of GDP- and GTP-tubulin measured by DSC was
found to be similar at free Mg2+ concentrations above 1 µM. However, it was dramatically reduced at the low
activity of Mg2+ ions of the EDTA containing buffer
employed for nucleotide exchange (7). This prompted an in-depth
examination of the system by means of the following complementary
biochemical and DSC experiments.
Binding of Mg2+ to GDP- and GTP-tubulin--
The
binding isotherms of Mg2+ to GDP- and GTP-tubulin in PEDTA
buffer at 10 °C were directly determined by high speed sedimentation of the protein (Fig. 1). The experimental
data for GDP-tubulin can be described assuming two classes of
independent binding sites in the 
-tubulin dimer: one
Mg2+ high affinity site (n1,Mg = 1.1 ± 0.2; K1,Mg = (1.1 ± 0.3) × 107 M
1), plus several low
affinity sites (n2,Mg = 48, K2,Mg = 106 M
1; the
latter values were taken from Frigon and Timasheff (17) and constrained
in the fitting procedure). Measurements of samples incubated for an
extra 1-2-h period at the lower ligand concentrations were essentially
identical, indicating equilibrium. High affinity Mg2+
binding to tubulin is reversible, since supplementing cation-depleted tubulin (equilibrated in 40 ± 5 nM free
Mg2+) to 360 ± 20 nM free Mg
2+ increased binding from 0.4 ± 0.1 to 0.75 ± 0.1 Mg2+ per tubulin heterodimer, which is within the
experimental error of the reference isotherm (Fig. 1).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1.
Binding of Mg2+ to GDP-tubulin
(solid circles) and GTP-tubulin (open circles).
Solid line, binding isotherm calculated assuming a simple
binding model (n1,Mg = 1.1 ± 0.2, K1,Mg = (1.1 ± 0.3) × 107
M 1 plus the low affinity sites
n2,Mg = 48, K2,Mg = 106 M 1 described by Frigon and Timasheff (17);
see the text for details). Dashed line, Mg2+
binding values obtained by adding one to the GDP-tubulin isotherm (solid line) in the interval indicated.
|
|
GTP-tubulin has one more Mg2+ binding site (Fig. 1,
empty symbols) than GDP-tubulin, with an
apparent affinity in the order of 105
M
1. The analysis of its Mg2+
binding isotherm is complicated by the presence of GDP, which has a
much higher affinity than GTP for the tubulin E-site at low
Mg2+concentration (3, 4). Therefore, as the cation
concentration decreases, GDP progressively exchanges into the
GTP-tubulin samples. GTP-tubulin in 63 µM free
Mg2+ had a measured nucleotide content (0.1 GDP and 1.7 GTP
per tubulin molecule), corresponding to a 90% of GTP-tubulin, and its
stoichiometry of binding of Mg2+ is one more cation than
that of GDP-tubulin. On the other hand, at 3.2 µM free
Mg2+ the binding stoichiometry is slightly higher than 1, but the nucleotide content (0.7 GDP and 1.2 GTP per tubulin molecule) indicates that only 25% of the protein is GTP-tubulin. The results are
compatible with the partial Mg2+ binding data of Mejillano
and Himes (23) in a different buffer.
These high affinity Mg2+ binding sites of tubulin, one in
GDP-tubulin and two in GTP-tubulin, have been previously identified as
the cation-binding loci at the N and E GTP-binding sites in
- and
-tubulin, respectively (3, 4, 24). However, binding isotherm of the
highest affinity Mg2+ to the N-site had not been measured;
nor had cation-depleted tubulin been studied.
Effect of Mg2+ Depletion on Nucleotide Release from
Tubulin--
Equilibration of tubulin in Mg2+ free buffers
results in a decrease in bound nucleotide, supposedly coming from
either nucleotide dissociation from the E-site or from the irreversible
protein denaturation which occurs for prolonged incubation times (4, 20). To know the role of the Mg2+ cation bound to the
N-site of GDP-tubulin in nucleotide binding, and to identify the sites
from which nucleotide may come off by cation removal, the GDP and GTP
bound to tubulin were determined as a function of the free
Mg2+ and incubation time (see Table
I for a summary). Tubulin samples with
the high affinity Mg2+ site saturated contain close to one
GTP and one GDP per 
-dimer. However, samples partially depleted
from the high affinity cation have their nucleotide content reduced to
0.6-0.7 GTP and 0.9 GDP per heterodimer at the conclusion of sample
preparation (an equivalent time of 0.5 h). Upon prolonged
incubation at 20 °C, the GTP (and GDP) stoichiometry decreased more
slowly, to values approaching the Mg2+/tubulin
stoichiometry of the samples (Table I). These results indicate that
dissociation of Mg2+ from the N-site (
-subunit), which
is quite reversible at short periods of time as shown above, results in
dissociation of GTP (and GDP), supposedly coming from the N-site (and
the E-site, respectively). This reveals the instability of the
cation-depleted tubulin. The results suggest the possibility that the
nucleotide binding capacity and, hence, the functionality of the
-subunit in the 
-dimer is controlled by the cation ligation
state of the
-subunit, and will be further addressed later.
GDP-tubulin equilibrated at 55 nM free Mg2+ and
re-equilibrated in 6 mM MgCl2 and 1 mM GTP polymerized in 3.4 M glycerol-containing buffer with a critical concentration (15 µM) 1.7 times
higher than that of a GTP-tubulin control directly equilibrated in 6 mM MgCl2 (9 µM). The slope of the
plot of plateau turbidity versus total protein concentration
was about 1.3 times lower than that of the control (data not shown).
Since GDP-tubulin is unable to assemble in Mg2+-glycerol
buffer (7), this implies that around 70 ± 10% of the
Mg2+-depleted protein has been able to back-exchange GTP
and reassemble. This result also suggests that nucleotide dissociation
during the time of cation depletion of tubulin results in an
irreversible conformational change, preventing the subsequent binding
of nucleotide to a fraction of
- and
-subunits.
The Kinetics of Tubulin Inactivation Depends on Mg2+
Activity--
The role of Mg2+ bound to the N-site on
tubulin inactivation at 20 °C was investigated monitoring the time
courses for the decay of paclitaxol-induced assembly, and for the
binding of the colchicine analogue MTC to GDP-tubulin, at different
free Mg2+ concentrations. GDP-tubulin equilibrated in 60 nM free Mg2+ initially retains more than 80%
of the corresponding activity at higher cation concentrations. However,
its inactivation is more rapid (half-life, t1/2 = 5 h; Fig. 2) than at 300 nM free Mg2+ (t1/2 = 7 h; data not shown), and much faster than at 100 µM free Mg2+ (estimated t1/2 ~ 47 h; Fig.
2), compatible with previous measurements of tubulin aging (20, 55).
For practical purposes, the kinetics after the initial decay can be
apparently described by first order reactions, whose rate constants,
k, decrease with the Mg2+ concentration
(4.0-5.5 × 10
5 s
1 at 60 nM, 2.5-3.0 × 10
5 s
1 at
300 nM, and 3.8-4.6 × 10
6 s
1 at 100 µM free
Mg2+).2 As a
control for sedimentation equilibrium measurements, the decay of MTC
binding by tubulin was also measured at 10 °C in 65 nM
free Mg2+, giving an apparent first order constant of
0.9 × 10
5 s
1 (t1/2 = 21 h; data not shown).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2.
Kinetics of GDP-tubulin inactivation at
20 °C, followed by either fluorescence of the MTC-tubulin complex
(circles) or Taxol®-induced microtubule
assembly (triangles). Closed symbols correspond
to samples equilibrated at ~0.1 mM free Mg2+,
whereas data in open symbols were taken at 60 nM
free Mg2+. The lines correspond to first order
kinetic rate constants of 4.6 × 10 6
s 1 (closed circles), 3.8 × 10 6 s 1 (closed triangles), and
4 × 10 5 s 1 (open
circles).
|
|
High Affinity Mg2+ Binding Enhances the Fluorescence of
MTC Bound to the Colchicine Site of Tubulin--
The addition of
Mg2+ to the complex of the colchicine analogue MTC with
tubulin, previously equilibrated at 50-60 nM free
Mg2+, leads in a few seconds to a large increase in the
fluorescence of the ligand (Fig. 3). The
variation is equivalent to the change in fluorescence observed upon MTC
binding to GDP-tubulin with its Mg2+ high affinity site
previously saturated with the cation. Control experiments indicated
that Mg2+ affects neither the negligible fluorescence of
unbound MTC nor the intrinsic (tryptophan) fluorescence of tubulin.
Furthermore, Mg2+-induced increase in the fluorescence of
the MTC-tubulin complex cannot be explained in terms of variation in
the extent of MTC binding to tubulin, since it was found to be
independent of the free Mg2+ concentration (0.7 ± 0.1 MTC/tubulin heterodimer). The apparent association constant of
Mg2+ to GDP-tubulin estimated from the MTC fluorescence
change (Fig. 3) was 9 × 106 M
1, essentially coincident
with the association constant of the high affinity Mg2+
(Fig. 1). Mg2+ binding also increased the fluorescence of
colchicine bound to tubulin, but only when the cation was bound prior
to the addition of colchicine (data not shown). This different
Mg2+ effect with MTC and colchicine might indicate that the
slow dissociation rate of the latter prevents the ligation of
Mg2+ to the N-site.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of Mg2+on the fluorescence
intensity of the tubulin-MTC complex (5 µM) at
20 °C. The line is a fluorescence titration curve calculated
for cation-binding to a single site (Fmax = 71, Fmin = 8, Kb = 9 × 106 M 1). Inset,
fluorescence spectra of the complex at 8 µM (solid
line) and 65 nM (dotted line) free
Mg2.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of Mg2+ on the far-UV CD
spectrum of GDP-tubulin (5 µM). Each spectrum
represents an average of four scans. Free Mg2+
concentrations are 1 mM (solid line), 3 µM (dotted line), and 65 nM
(dashed line).
|
|
The simplest interpretation of these results is that the
microenvironment of tubulin-bound MTC is sensitive to the high affinity bound Mg2+ ion, which actually induces the fluorescence of
this colchicine site probe. Furthermore, the results also reveal
communication between tubulin subunits, since the binding of the cation
to N-site in
-tubulin modifies the properties of the colchicine
site, whose locus is at the
-subunit, possibly near the

-subunit interface (56).
Modification of tubulin secondary structure upon removal of the high
affinity bound Mg2+ was checked by CD spectroscopy.
Equilibration of the protein in 65 nM free Mg2+
leads to a small reduction in the absolute magnitude of the dichroic signal at 210-220 nm (Fig. 4). The effect is independent of having GDP
or GTP in the buffer, suggesting that the observed change is in part
induced by Mg2+ coordination at the N-site. The analysis of
this small change in the CD spectrum of tubulin indicated very small
differences in the estimated secondary structure content. The CD change
can be partially reversed by increasing the free Mg2+
concentration up to 1 mM (data not shown). Preliminary CD
kinetic experiments of Mg2+ dissociation from tubulin and
their subsequent reassociation, indicated that dissociation is slow (on
the order of minutes), whereas reassociation is comparatively fast (on
the order of seconds).
Role of Mg2+ in 
-Tubulin
Association--
Cations bound with high affinity to oligomeric
proteins frequently have structural roles, and their removal is linked
to a marked weakening of protein-protein association equilibria (two examples are the platelet integrin
IIb
3
(57) and the complement C1 subcomponent (48)). For this reason, the
influence of Mg2+ (low and high affinity binding sites) on
the dimerization equilibrium of tubulin was analyzed by analytical
ultracentrifugation. GDP-tubulin equilibrated in 50-60 nM
Mg2+, at an initial protein concentration of 15 µM, has the same sedimentation coefficient
(s20,w = 5.8 ± 0.2 S) and relative
molecular mass (109,000 ± 8,000; Fig.
5) as the intact 
-tubulin dimer. However, the tubulin dimer dissociation is patent at lower
concentrations, as was analyzed by sedimentation equilibrium at
10 °C. Fig. 6 shows the variation in
the average molecular mass of tubulin as a function of total tubulin
and free Mg2+ concentrations. The results indicate that
removal of Mg2+ from its high affinity site increases
dissociation of the tubulin dimer. This behavior reflects the linkage
of Mg2+ binding and tubulin self-association equilibria.
Lowering the free Mg2+ concentration reduced by an order of
magnitude the apparent equilibrium dimerization constant of

-tubulin (K2), from ~107
M
1 at ~100 µM free cation, to
4 × 106 M
1 at 1-2
µM cation, and to 1.6 × 106
M
1 at 50 nM free cation (see
Table II and Fig. 6).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
Analytical ultracentrifugation of GDP-tubulin
at 50-60 nM free Mg2+ (10 °C).
Sedimentation equilibrium profile of tubulin under the conditions used
in DSC experiments (15 µM loading protein concentration).
Inset, sedimentation velocity distribution of tubulin
samples. The solid line corresponds to the same tubulin showed in the main figure. The dashed and dotted
lines are GDP-tubulin after 30 min at 40 °C and 60 °C,
respectively.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Influence of Mg2+on the
GDP-tubulin dimerization equilibrium. Dependence of the apparent
weight-average molecular mass of GDP-tubulin on free Mg2+
and protein concentration. Cation concentrations: 50 µM
(closed circles), 1.6 µM (open
circles), 60 nM (solid triangles). The solid lines were calculated for a monomer-dimer equilibrium
model using the equilibrium constants given in Table II. For
illustrative purposes, the Mr of - and -tubulin
(55,000), as well as the value for the heterodimer (2 × 55,000)
are indicated by the dashed and dotted lines,
respectively. Inset, sedimentation equilibrium gradients of
tubulin (loading protein concentrations: 1, 2.5, and 5 µM; 26,000 rpm and 10 °C) at 60 nM free
magnesium. The solid lines represent the best-fit function
(see Table II).
|
|
The simplest interpretation of these results is that both the high
affinity binding of one Mg2+ ion to the N-site and the
binding of lower affinity cations stabilize the tubulin heterodimer.
The data are compatible with the results obtained in the
10
3 to 10
4 M free
Mg2+ concentration range by Shearwin et al. (8),
who suggested the involvement of two weakly bound Mg2+ ions
in the association of the GDP-tubulin heterodimer. A linked equilibria
analysis of our data supports the notion that both low and high
affinity Mg2+ enhance tubulin dimerization. As shown under
"," the combined sedimentation equilibria data
may be reasonably accounted for by a
Mg2+-dependent dimerization model, which is
compatible with both the experimental dependence of
K2 on the cation concentration (Table II) and
the Mg2+-binding isotherm at high protein concentration
(Fig. 1). According to this model, only one of the isolated subunits of
GDP-tubulin bears a high affinity Mg2+ binding site, with
an intrinsic binding constant of 2 × 106
M
1, whereas the heterodimer has two
independent Mg2+ binding sites, with binding constants
1 × 107 M
1 and 6 × 104 M
1, respectively. The
estimated value for the intrinsic dimerization constant of tubulin in
the absence of Mg2+ is
K20 = 106
M
1. The limited dissociation range of tubulin
at the lowest protein concentration that could be measured in the
analytical ultracentrifuge, as well as the need for avoiding the
possible influence of tubulin denaturation processes at longer
equilibrium times, preclude a more complete quantitative analysis in
terms of linked functions (58, 59).
Roles of Mg2+ and Nucleotide in the Thermal Stability
of Tubulin--
The influence of Mg2+ on the thermal
stability of tubulin was analyzed by differential scanning calorimetry.
Fig. 7A compares representative DSC profiles of GDP-tubulin in PEDTA buffer at increasing free Mg2+ concentrations. The experimental
curves show that tubulin is strongly stabilized against thermally
induced denaturation through interaction with the cation in the 10 nM to ~1 µM range of free ligand
concentration. Above 1 µM free Mg2+, the heat
capacity curve presents a single asymmetric peak with an enthalpy
change of 180 ± 10 kcal mol
1 and a
Tm of 55 °C, whereas at the lower cation
concentrations (25-40 nM free Mg2+) the
denaturation enthalpy change decreases to 130 ± 25 kcal mol
1, and the endotherm tends to become separated into
two peaks (Figs. 7A and 9B), the main one with a
Tm of 46 °C at 25 nM free
Mg2+. The origin of the two peaks will be analyzed
later.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 7.
Panel A, thermal denaturation curves of
GDP-tubulin (15 µM) at increasing concentrations of
Mg2+. The experiments were done at a scan rate of 0.5 K·min 1. Free Mg2+ concentrations (from top
to bottom): 280 µM, 4 µM, 920 nM, 500 nM, 182 nM, and 50 nM. Panel B, dependence of denaturation
Tm values of GDP-tubulin (15 µM) on
the free Mg2+ concentration (solid circles);
open circles correspond to GTP-tubulin.
|
|
Substitution of GDP by GTP at the E-site does not modify tubulin
stabilization by Mg2+, which nearly reaches a plateau above
1 µM free Mg2+ (Fig. 7B). The
results strongly suggest that the cation responsible for the
substantial tubulin stabilization observed is the Mg2+ ion
coordinated with the nucleotide N-site, since (i) the stabilization essentially coincides with the binding of this high affinity cation, (ii) the additional cation bound by GTP-tubulin has insignificant effect on the thermal stability of the protein, and (iii) the affinity
of Mg2+ for the E-site in GDP-tubulin is known to be about
103 times lower than in GTP-tubulin (3, 4). Table
III summarizes the parameters measured
for the thermal denaturation of tubulin.
View this table:
[in this window]
[in a new window]
|
Table III
Thermal denaturation data of GDP- and GTP-tubulin at different free
Mg2+ concentrations and scanning rates
|
|
The reversal of tubulin destabilization induced by Mg2+
depletion was checked by preparing protein samples equilibrated at
different cation concentrations and then adding Mg2+ up to
saturation (Table IV). The
destabilization induced by the cation dissociation was 95% reversible
in tubulin samples initially equilibrated in 180 nM free
Mg2+ and immediately supplemented with 280 µM
free Mg2+ (
Hd = 176 kcal
mol
1, Tm = 56.1 °C; Fig.
8, curve b). However, when
Mg2+ was added after 2 h of incubation at 20 °C in
the equilibration buffer, the shape of the calorimetric profile was
indistinguishable from that obtained upon saturation with the cation
immediately after protein elution, but the enthalpy change dropped to
about 75% of the initial value (
Hd = 139 kcal·mol
1; T = 56.1 °C;
curve c in Fig. 8).The drop in
Hd
observed after 2 h at 20 °C correlates with the value expected
from the kinetics of tubulin inactivation under same conditions (Fig.
2). Reconstitution of tubulin samples initially prepared in 40 nM free Mg2+ (
Mg = 0.4,
GTP = 0.65) results in heat capacity denaturation curves
with the same Tm value as in the control experiments but with a lower enthalpy change (142 kcal·mol
1;
curve e, Fig. 8). The percentage of reversibility obtained
by saturation with Mg2+ immediately after preequilibration
in Mg2+-depleted buffers, correlates well with the initial
GTP/tubulin stoichiometry of the samples. These results indicate that
Mg2+- or nucleotide-depleted tubulin slowly evolves in an
irreversible way toward a state that does not undergo a temperature
induced cooperative transition, and that GTP bound to the N-site of
tubulin might determine the reversibility of Mg2+
dissociation. Addition of colchicine stabilized the cation-depleted tubulin, similarly to addition of Mg2+, whereas the
reversible binding of the colchicine analogue MTC had a much weaker
effect (Table V).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 8.
Reversibility of the Mg2+
stabilizing effect on thermal denaturation of GDP-tubulin (15 µM). Curve a, endotherm of tubulin equilibrated at 180 µM free Mg2+; curve
b, the same as a but supplemented with Mg2+
(280 µM final free concentration) immediately after
column elution; curve c, the same as a but
supplemented with Mg2+ after incubation for 2 h at
20 °C (scan rate 30 °C·h 1); curve d,
endotherm of GDP-tubulin equilibrated at 45 nM free Mg2+; curve e, the same as d, but
supplemented up to 470 µM free Mg2+
immediately after elution from the preparative column (scan rate 45 °C·h 1).
|
|
Kinetic Control of the Thermal Denaturation of Tubulin by High
Affinity Mg2+ Binding: Mechanism of Thermal
Denaturation--
Reheating of tubulin samples cooled after the first
thermal scan showed that thermal denaturation of tubulin is
irreversible under all the conditions tested. The thermograms depend on
the scan rate (see Fig. 9, A
and B) and the analysis of DSC curves showed that, on
saturation of the high affinity Mg2+ binding site, the
variation in the excess heat capacity with temperature follows the
behavior predicted by the two-state kinetic model (53, 54). Fig.
9C shows the temperature dependence of the apparent
denaturation rate constant at different free
Mg2+concentrations, calculated according to this model.
This result means that only Mg2+ liganded (N-site) and
denatured tubulin are significantly populated within the denaturation
temperature range. A good correlation was found between the kinetic
constants of inactivation calculated from the rate of CD change at
55 °C (
220, free [Mg2+] = 180 nM) or from DSC data at the same temperature and cation concentration (3 × 10
3 s
1 and
3.5 × 10
3 s
1, respectively). However,
the kinetic constants extrapolated from the DSC data to 20 °C are
several orders of magnitude smaller than those measured from tubulin
inactivation at this temperature, suggesting a different origin for the
two processes in the lower temperature range.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 9.
Panels A and B, DSC traces of
GDP-tubulin (15 µM) at different scanning rates and free
Mg2+ concentrations. Panel A, 280 µM free Mg2+ (30 and
20 °C·h 1; top to bottom). Panel B, 40 nM free Mg2+ (60, 45, and
30 °C·h 1; from top to bottom). Panel C,
Arrhenius plot of the kinetic rate constant,
kapp, derived from calorimetric data assuming
the two-state kinetic model, at different free Mg2+
concentrations: 280 µM (diamonds), 927 nM (circles), 500 nM
(triangles), and 182 nM
(squares).
|
|
At the lowest free Mg2+concentrations (40-100
nM), the denaturation process becomes complex as indicated
by the presence of a shoulder or small peak in the low temperature side
(Figs. 7-9). This is also evident in the loss of secondary structure,
monitored by CD at 220 nm (Fig. 10).The
enthalpy change associated with the low temperature shoulder can be
roughly estimated to be ~30 ± 5 kcal·mol
1. The
CD spectra of thermally denatured tubulin has residual
sheet
secondary structure (60). The apparent biphasic denaturation of tubulin
at subsaturating levels of Mg2+ could be generated by
different processes such as kinetic stabilization of unfolding
intermediates, uncoupling of
- and
-subunit denaturation, or
association of the denatured state. In addition, given the slow
dissociation of the nucleotide from tubulin and the presence of GTP at
substoichiometric ratios under these conditions (61-63), the low and
high temperature peaks could also derive from the unligated and
nucleotide-bound tubulin, respectively.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 10.
Dependence of the excess enthalpy function
(Hexcess) and the CD signal at 220 nm
( 220) of tubulin with temperature (65 nM
free Mg2+). The solid line shows the excess
enthalpy values of the corresponding DSC curve. The symbols
show the experimental CD data. Tubulin concentration was 15 µM and the temperature scan rate 0.5 °C
min 1.
|
|
Sedimentation velocity measurements have shown that the association
state of thermally denatured tubulin depends on the Mg2+
concentration. Incubation of GDP-tubulin equilibrated in 50 nM free Mg2+ at 40 °C for 30 min induces a
partial aggregation of tubulin, giving a bimodal sedimentation velocity
profile, in which approximately half of the protein sediments as the
tubulin dimer (s20,w = 5.7 S) and the
other half as a 12 S oligomer (see dotted line in the
inset of Fig. 5). Furthermore, 30-min incubation at 60 °C results in a higher percentage (~70%) of tubulin aggregation (13-14 S). However, when the same treatment was performed on GDP-tubulin with
the high affinity site occupied by Mg2+, aggregation was
not evident (s20,w = 5.9 S). These results suggest that tubulin aggregation might be involved in the
generation of the biphasic denaturation curves. Nevertheless, contributions from other processes (see above) cannot be ruled out. A
possible minimal scheme to account for thermal denaturation of tubulin
at the Mg2+ concentration range explored is as follows.
KGTP
and
KMg
are the GTP and
Mg2+ binding constants to N-site, k3
is the denaturation rate constant of tubulin·GTP·Mg, and k1 and k2 are the
limiting rate constants for denaturation of N-site unliganded and
GTP-bound tubulin, respectively; Ii are irreversibly
denatured state(s) of tubulin (I1 and
I2 are 12-14 S aggregated species, and
I3 is 5.9 S denatured tubulin) and
Xi indicates the possibility of intermediate steps during denaturation.
 |
CONCLUSION |
The results reported in this study provide new insights into
tubulin structure and function. This is schematically summarized in
Fig. 11. The nucleotide
-phosphate
and the coordinated Mg2+ ion at the E-site (
) of
tubulin, which regulate the tubulin assembly function and microtubule
stability, have practically undetectable effects on the stability and
on most of the solution properties of the 
-tubulin dimer.
However, one high affinity Mg2+ ion, bound to a site
identified as the non-functional nucleotide N-site (
), has profound
kinetic and thermal stabilizing effects, and induces the association of
the 
-dimer. The
- and
-subunits seem to communicate with
each other; the binding of Mg2+ to the N-site in the
-subunit induces the fluorescence of a probe bound to the colchicine
site in the
-subunit, and colchicine binding thermally stabilizes
Mg2+-depleted tubulin. These properties are most simply
explained by proposing that both the colchicine site (
) (56) and the N-site Mg2+ (
) (this study) are located at the 
dimerization interface. It follows from subunit homology that the
functional E-site (in
) should be at the longitudinal dimer-dimer
interface leading to protofilament formation (64), consistent with the
activation of tubulin GTPase in linear oligomers (65).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 11.
Model scheme proposed to explain and
summarize the results of this work. The nucleotide and
Mg2+ bound to the E-site, which are known to regulate the
tubulin assembly function, have insignificant effects on the stability of tubulin. The high affinity Mg2+ ion bound at the
nucleotide N-site controls the stability of the  -tubulin dimer.
The simplest explanation for the observed effect of the
Mg2+ binding at the N-site on the fluorescence of a probe
bound to the colchicine site (COL) is close communication,
i.e. both sites being near the - dimerization
interface. It follows that nucleotide-Mg2+ E-site should be
at the interface of association of the dimer with the next dimer along
one protofilament of the microtubule. The dashed arrow from
the colchicine site to E-site indicates the allosteric communication,
which activates the GTPase activity in the  -dimer upon colchicine
binding, although the sites are more than 2.4 nm apart (for this and
other distances, see Ref. 66). The shape of the tubulin dimer
corresponds to a contour view from the outside of a low resolution
microtubule model deduced from x-ray solution scattering (67).
|
|
All tubulins probably evolved from a common nucleotide-binding
ancestor. The GTP and Mg2+ binding functionalities were
made essential for the maintenance of the protein stability in
-tubulin, whereas
-tubulin acquired the capability to hydrolyze
bound GTP upon activation by proper contact with other tubulin
molecules, which is the basic mechanism controlling microtubule
stability. The sites of binding of the antimitotic drugs colchicine,
vinblastine, and paclitaxel, for which endogenous ligands are unknown,
are also primarily located in
-tubulin. It is presently unclear how
a dimer was selected to assemble microtubules.