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INTRODUCTION |
The Op18/stathmin family of microtubule regulators includes the
ubiquitous cytosolic Op18/stathmin
(Op18)1 and the neuronal
proteins RB3, SCG10, and SCLIP (for review, see Ref. 1). A
characteristic feature of the neural family members is a stretch of
hydrophobic amino acids at the N terminus, which includes two Cys
residues that serve as palmitoylation sites and mediate association to
intracellular membranes, primarily at the Golgi apparatus (for review,
see Ref. 2). The ubiquitous Op18 protein, which lacks this stretch of
hydrophobic amino acids, has a widespread distribution in the cytosol.
Op18/stathmin family members form ternary complexes with two tubulin
heterodimers, and the overall shape of the complex has been revealed by
transmission electron microscopy and a low resolution x-ray structure
(3, 4). The complex can be described as two tubulin heterodimers in a
slightly curved head-to-tail alignment, with each one of the tandem
helical repeats of Op18 binding along one heterodimer to generate a
ternary tandem tubulin heterodimer complex, which is stabilized by
longitudinal interactions between the two heterodimers (see Fig. 1). As
predicted by these stabilizing heterodimer-heterodimer interactions
within the complex, Op18 binds two heterodimers according to a two-site
positive cooperative model, which strongly favors formation of a
ternary complex rather than binary complex formation with a single
tubulin heterodimer (5). The 4-Å resolution x-ray structure has
allowed rejection of alternative models for the ternary complex (6, 7),
but was of insufficient resolution to resolve the orientation of the extended Op18 helix. Cross-linking experiments, however, indicate that
the N terminus of Op18 is orientated toward the exposed
-tubulin end
of the head-to-tail-aligned heterodimers (8) (see Fig. 1).
Microtubules are polymers built up from
/
-tubulin heterodimers,
and a characteristic feature of individual microtubules is frequent and
stochastic switching between polymerization and depolymerization
cycles, a phenomenon termed dynamic instability (for review, see Ref.
9). The
-tubulin subunit of the heterodimer contains an exchangeable
GTP-binding site (termed E-site), and microtubules utilize
polymerization-induced GTP hydrolysis to generate dynamic instability.
The mechanism of GTP hydrolysis involves longitudinal interactions of
polymerizing
/
heterodimers via a catalytic loop located on the
-tubulin that interacts with the E-site of the adjacent
-tubulin
subunit (10). The tip of a polymerizing microtubule contains a
stabilizing cap of GTP-tubulin, the loss of which results in a
catastrophe (i.e. transition from a growing to a shrinking polymer).
Op18 was originally described as being a specific catastrophe-promoting
protein (11), but this was subsequently challenged by a study claiming
that Op18 acts solely by sequestering tubulin heterodimers (12, 13).
This controversy was resolved by a study demonstrating that Op18
mediates both catastrophe promotion and sequestration of tubulin and
that detection of these activities depends on buffer conditions (14).
It was also shown in the same study that the non-helical N-terminal
region is essential for catastrophe-promoting activity, whereas the
extended helical part of Op18 containing the two imperfect repeats is
necessary and sufficient for tubulin-sequestering activity. Recent
studies involving ectopic expression of truncated/mutated Op18
derivatives in human cell lines have indicated the significance of the
functional dichotomy of Op18 that is observed in vitro (5,
15, 16).
How Op18 promotes catastrophes is still obscure, but given that the
second helical repeat is dispensable, binding studies (5, 7) combined
with recent structural insights (4) allow the conclusion that the
mechanism does not involve the two-site positive cooperative binding
activity of Op18 that is required for efficient formation of ternary
tandem tubulin heterodimer complexes (see Fig. 1). Moreover, such
recent structural insights readily explain the requirement for the
second helical repeat in generating an Op18·tubulin
heterodimer complex of sufficient stability to exert
tubulin-sequestering activity. The two-site positive cooperative
binding activity of Op18 also has additional functional consequences
that are distinct from tubulin sequestration, such as inhibition of GTP
exchange and stimulation of autonomous low rate GTP hydrolysis within
the ternary complex (5, 7, 17). Truncation of the second helical repeat
of Op18, which does not alter the catastrophe-promoting activity,
terminates stimulation of GTP hydrolysis (5, 7). Hence, stimulation of
tubulin GTP hydrolysis by Op18 depends strictly on the cooperative tubulin heterodimer binding activity that mediates the tubulin head-to-tail configuration of the complex.
As outlined above, the cooperative Op18 binding of two tubulin
heterodimers inhibits GTP exchange and stimulates autonomous low rate
GTP hydrolysis within the ternary complex. In the present study, we
have functionally dissected Op18 and the homologous RB3 and SCG10
proteins with the aim of understanding the significance of these
GTP-regulatory events.
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MATERIALS AND METHODS |
DNA Constructs, Expression and Purification of Recombinant
Proteins--
Wild-type Op18 and an Op18 derivative with the
non-helical N-terminal 45 amino acids deleted (termed Op18-R1 + 2 because it contains the tandem repeats 1 and 2 of the extended helix;
see Fig. 1) have been described (5). Both of these Op18 derivatives contained an eight-amino acid C-terminal FLAG epitope. Soluble SCG10
(sSCG10), with the hydrophobic N-terminal 30-amino acid membrane
targeting region removed, was prepared by a PCR strategy using a
full-length cDNA clone of SCG10 (SCG10-8.6, (18), a gift from Dr.
N. Mori) as a template. The eight-amino acid C-terminal FLAG epitope
was introduced by a 24-nucleotide insertion before the stop codon in
the 3' PCR primer. An N-terminally truncated SCG10 derivative (termed
SCG10-R1 + 2), which corresponded to Op18-R1 + 2 (see Fig. 1), was
prepared by a PCR strategy using sSCG10 DNA as a template. As part of
the cloning strategy for truncated proteins, the first three amino
acids of the N terminus of Op18 were added to the N terminus of Op18-R1 + 2 and SCG10-R1 + 2 derivatives. The soluble form of RB3 (sRB3) and
the RB3-R1 + 2 derivative were constructed using an identical PCR
strategy to that described above for SCG10. An RB3 cDNA, obtained
from Research Genetics, Huntsville, AL, was used as a template
(GenBankTM accession number AL534520). The Op18-family
derivatives described above were expressed with a six-residue His tag
at the N terminus and purified from Escherichia coli using
pET-3d expression as described previously (5). Construction of the
Op18-pmut1, where the codons for Leu-47, Ile-50, and Leu-54 are
exchanged for Ala codons, has been described previously (originally
termed Op18-cc m1) (19). Op18-pmut2, where the codons for Val-68,
Leu-69, and Leu-72 are exchanged for Ala codons, and Op18-pmut3, where
the codons for Val-82 and Leu-83 are exchanged for Ala codons, were constructed using a general strategy where mutations were introduced into subfragments of the coding region by overlapping PCR using wild-type Op18 as template. Coding regions of these Op18 derivatives were expressed and purified as glutathione S-transferase
(GST) fusion proteins in pGEX 4T-1 (7). The coding sequences of
PCR-generated fragments were confirmed by nucleotide sequence analysis
using the ABI PRISM dye terminator cycle sequencing kit from
PerkinElmer Life Sciences. Purified recombinant proteins were routinely
analyzed by SDS-PAGE to confirm the homogeneity and determine the
protein concentration of each preparation.
Assays of Tubulin GTPase Activity--
Analysis of tubulin
GTPase activity was performed in PEM buffer (80 mM
piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM
EGTA, 4 mM Mg2+) containing 17% glycerol
adjusted to the indicated pH with NaOH. The buffer also contained 5 mM adenyl-5'-yl imidodiphosphate (AMP-PNP; to inhibit
nonspecific ATPase activity) as described previously (7). In brief,
tubulin (TL238, Cytoskeleton, Inc.) was incubated with
[
-32P]GTP, the resulting
tubulin·[
-32P]GTP complexes were recovered by
centrifugation through a desalting column (P-30 Micro Bio-Spin,
Bio-Rad), and single-turnover GTP hydrolysis was followed at 37 °C.
Control experiments showed that the Op18 preparations used neither bind
nor hydrolyze [
-32P]GTP or cause dissociation of
tubulin-bound [
-32P]GTP. Nucleotide hydrolysis was
quantified by ascending chromatography as described (7), which allows
reproducible analysis of less than 0.2% hydrolysis of the
[
-32P]GTP.
Determination of Tubulin-GTP Exchange Rates and Plasmon Resonance
Experiments--
[
-32P]GTP-tubulin (0.8 mol GTP/mol
tubulin, ~4 × 1015 dpm/mol) was incubated at 5 µM (in PEM buffer at the stated pH and glycerol concentration) on ice for 10 min in the presence or absence of the
indicated Op18-family derivative. The reaction was started by the
addition of 2 mM cold GTP at 37 °C, and exchange rates were followed over time by separation of unbound
[
-32P]GTP through a desalting column (P-30 Micro
Bio-Spin, Bio-Rad). These columns retained more than 99.99% of all
non-tubulin bound [
-32P]GTP, whereas the flow-through
contained tubulin and Op18 (>95% yield). Twenty microliters of the
reaction mixture was sampled per time point. The GTP exchange rates
were calculated by quantifying the radioactivity of the samples and the
desalting columns. Plasmon resonance competition experiments were
carried out on a BIAcore 3000 system with Op18 immobilized by amine
coupling on a CM5 chip according to the instructions of the
manufacturer. Analyses were performed with the indicated concentrations
of tubulin in PEM, pH 6.8, premixed with graded concentrations of Op18
derivatives. The free tubulin concentrations were determined from the
plateau levels by comparison with a standard tubulin curve, as
described in the BIAcore handbook. Dissociation constants refer to the
binding of two tubulin heterodimers.
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RESULTS |
Modulation of Tubulin GTP Exchange and Hydrolysis by Complex
Formation with Op18/Stathmin Family Members--
Complex
formation between Op18 and tubulin heterodimers has previously been
shown to result in low rate tubulin GTP hydrolysis (5). To determine
whether this property is conserved, we analyzed soluble derivatives of
two neural members of the Op18/stathmin family, SCG10 and RB3, in which
the membrane-targeting regions were deleted (see sSCG10 and sRB3 in
Fig. 1). Single-turnover tubulin GTP
hydrolysis was determined at 37 °C by incubation of Op18/stathmin
family derivatives with tubulin at a concentration that was
sufficiently low to avoid polymerization. As shown in Fig.
2, all three Op18/stathmin family members
tested stimulated tubulin GTP hydrolysis ~3-fold as compared with the
basal hydrolysis rate. Moreover, analysis of N-terminally truncated
derivatives that only encompass repeats 1 and 2 (see Fig. 1) revealed
unaltered tubulin GTPase stimulation (compare Fig. 2, A and
B). It should be noted that truncation of the 45 residues at
the non-helical N terminus resulted in a shift in dose-response in all
cases, which indicates decreased tubulin heterodimer binding affinity. However, under the present assay conditions, each derivative was used
at a concentration that was sufficient to reach the plateau level of
tubulin GTPase stimulation (data not shown). Hence, the level of GTPase
stimulation appears to be completely conserved among Op18/stathmin
family members and requires only the region shown to be directly
involved in the cooperative tubulin heterodimer binding activity, which
generates the ternary tandem tubulin heterodimer complex (see Fig.
1).

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Fig. 1.
Schematic representation of Op18, SCG10, and
RB3 family members and their derivatives used in this study. At
the top, native Op18 is depicted with a non-helical
N-terminal region, which appears to have a low degree of secondary
structure (residues 1-45) (6) and an extended -helical region
containing two low-homology repeats, designated Repeat
1 and Repeat 2, which are separated by
51 residues (4). The positions of the two longitudinally arranged
tubulin heterodimers along the two helical repeats are depicted
according to the low resolution x-ray structure (4), with the
orientation of the N terminus toward the -tubulin end of the ternary
tandem tubulin heterodimer complex as indicated by cross-linking
experiments (8). Native SCG10 and RB3 have stretches of hydrophobic
residues at their N terminus, which are responsible for membrane
targeting (black boxes). These hydrophobic
stretches were deleted to produce soluble derivatives that contain only
the Op18/stathmin homology region (sSCG10 and sRB3, respectively).
Derivatives with intact repeat 1 and 2 but with the non-helical
N-terminal region truncated are also depicted (Op18-R1 + 2, SCG10-R1 + 2, and RB3-R1 + 2).
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Fig. 2.
Stimulation of single-turnover tubulin GTP
hydrolysis and tubulin GTP exchange inhibition at 37 °C.
A and B, the initial rates of single-turnover
tubulin GTP hydrolysis were evaluated in the absence and presence of
the indicated derivatives (see Fig. 1) at 37 °C in PEM buffer, pH
6.8, containing 17% glycerol. Op18/stathmin-family derivatives were
used at 20 µM together with 5 µM tubulin
heterodimers, which is sufficient for attainment of plateau levels of
activity (data not shown). C and D, GTP exchange
rates of tubulin were determined under the conditions used for
determination of GTP exchange rates. The exchange rates were calculated
by fitting data points to a hyperbola using the one-phase exponential
association/dissociation equations provided by GraphPad Prism. Assays
for GTP exchange and GTP hydrolysis employed tubulin heterodimers in a
1:1 molar complex with [ -32P]GTP and were performed as
described under "Materials and Methods."
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Op18 has also been shown previously to inhibit tubulin GTP exchange (5,
17). A comparison of tubulin GTP exchange inhibitory properties of the
three Op18/stathmin family members is shown in Fig. 2C, and
the exchange rates calculated were as follows: control, 0.82 min
1; Op18, 0.061 min
1; sSCG10, 0.036 min
1; and sRB3, 0.0076 min
1. It is evident
from these results that, under identical conditions to those used for
tubulin GTPase assays, Op18 causes inhibition of the tubulin GTP
exchange rate by a factor of ~10. Interestingly, the effects of
sSCG10 and sRB3 were even more potent, and the observed GTP exchange
inhibition lies in the order sRB3 > sSCG10 > Op18. This is
the same order as the order reported for stability of the ternary
complexes between tubulin heterodimers and these Op18/stathmin family
members (20), which has been confirmed by us subsequently (data not
shown). Hence, the stability of the ternary tandem tubulin heterodimer
complex appears to be an important determinant for tubulin GTP exchange inhibition.
The 45-residue non-helical N terminus of Op18 contributes to tubulin
GTP exchange inhibition at 37 °C (19). The cognate N-terminal
regions of SCG10 and RB3 act similarly, as evidenced by decreased
tubulin GTP exchange inhibition by the SCG10-R1 + 2 and RB3-R1 + 2 derivatives that only encompass the extended helical region (Fig.
2D) (exchange rates were as follows: Op18-R1 + 2, 0.14 min
1; SCG10-R1 + 2, 0.083 min
1; and RB3-R1 + 2, 0,054 min
1). By comparing the data in Fig. 2,
C and D, however, it is clear that the
N-terminally truncated derivatives still retain substantial GTP
exchange inhibitory activity, with RB3-R1 + 2 being as potent as
full-length Op18. It is also clear from Fig. 2, C and
D that the relative potency of each of the N-terminally
truncated derivatives lies in the same order as the non-truncated
derivatives, i.e. sRB3 > sSCG10 > Op18.
To further address the structural requirements underlying tubulin GTP
exchange rates, assays were performed on ice to increase the stability
of complexes. We used the same glycerol-containing buffer as in the
experiments described above, because glycerol greatly increases the
tubulin heterodimer binding affinity of both full-length and truncated
Op18 derivatives, thereby facilitating saturated binding (19). To
estimate the affinity of tubulin heterodimer binding under these
conditions, plasmon resonance competition experiments were performed
(Fig. 3A). The data show that
truncation of the 45-residue non-helical N-terminal region results in a
significant decrease in binding affinity toward tubulin heterodimers.
From these data, the free tubulin concentrations at half-saturation
(i.e. the apparent dissociation constant, ignoring two-site
binding cooperativity) were calculated to be <0.2 µM for
Op18 and 2.5 µM for Op18-R1 + 2. Analysis of the tubulin
GTP exchange inhibitory properties of these two Op18 derivatives
reveals similar levels of GTP exchange inhibitory activities if
determined at 0 °C (Fig. 3B), and the calculated exchange
rates were 0.012 min
1 for Op18 and 0.015 min
1 for Op18-R1 + 2 as compared with 0.16 min
1 for the control. These data also show that the
potency of GTP exchange inhibition is dramatically increased at 0 °C
compared with 37 °C (compare Figs. 2C and 3B).
However, this can be attributed in part to the fact that the GTP
exchange rate of pure tubulin is reduced at 0 °C in a
glycerol-containing buffer, which has also been observed by others
(21).

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Fig. 3.
Tubulin GTP exchange inhibition at
0 °C. A, Plasmon resonance competition experiments
at 4 °C using PEM buffer, pH 6.8, in the presence of 10% glycerol.
Graded concentrations of the Op18 derivative indicated were mixed with
5 µM tubulin heterodimers. The dotted
line depicts complete complex formation at a 1:2 molar ratio
of Op18/tubulin heterodimers. B, the initial rates of
single-turnover tubulin GTP hydrolysis were evaluated at 0 °C in the
absence and presence of the indicated derivatives using the conditions
described in the Fig. 2 legend.
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It seems clear from the data in Fig. 3 that the non-helical N-terminal
region of Op18 is not a structural requirement for potent inhibition of
GTP exchange. It follows that the enhancing effect of the N-terminal
region on GTP exchange inhibition observed at 37 °C (Fig. 2) is an
indirect effect mediated by the affinity-enhancing activity of this
non-helical part of Op18/stathmin family members (Fig. 3A).
It is also notable that the slopes of GTP exchange both at 37 °C
(Fig. 2) and at 0 °C (Fig. 3) are mono-phasic, which suggests
similar GTP exchange rates at both E-sites contained within the ternary
complex. The solved x-ray structure shows that repeats 1 and 2 of
Op18/stathmin family proteins are not in contact with the exposed
E-site at one end of the complex, whereas the E-site that is interfaced
between the two heterodimers can be predicted to be enclosed (Ref. 4;
see Fig. 1). Thus, it seems that the cooperative tubulin
heterodimer-binding activity of Op18-like proteins inhibits GTP
exchange at the exposed E-site by an allosteric effect.
Stoichiometry of GTP Hydrolysis within the Ternary Tandem Tubulin
Heterodimer Complex--
To address whether GTP is hydrolyzed at one
or both of the E-sites of the two head-to-tail aligned tubulin
heterodimers, we performed kinetic analysis of GTP/GDP conversion
within the ternary complex. However, as indicated by data in Fig. 2,
the rate of GTP hydrolysis in PEM buffer is too low relative to the
half-life of the complex to obtain conclusive data by this approach.
Therefore, nocodazole was employed to increase the rate of hydrolysis,
because this drug has been reported to enhance both basal tubulin
GTPase activity (22) and the GTPase activity stimulated by Op18 (5). The addition of nocodazole results in a uniform ~5-fold increase in
the GTP hydrolysis rate of tubulin heterodimers in complex with Op18,
SCG10, or RB3 (data not shown).
Kinetic analysis of nocodazole enhanced [
-32P]GTP
hydrolysis within ternary complexes generated using sRB3, which is the
complex with the longest half-life of GTP exchange (see Fig.
2C), suggesting that only about half of the tubulin-bound
GTP is susceptible to hydrolysis (Fig.
4A). Thus, in the presence of
excess cold GTP, which restricts detection of hydrolysis to those
complexes formed prior to the initiation of the time course, hydrolysis
of labeled GTP approaches a plateau level at ~50%. This is below the
level of hydrolysis observed under conditions allowing multiple rounds of [
-32P]GTP binding, i.e. in the absence
of cold GTP. Interpretation of the experiment shown in Fig.
4A is somewhat complicated by the fact that GTP/GDP ratios
were determined in the total assay mix, and this includes
[
-32P]GTP/GDP, which may dissociate from tubulin
during the time-course. To ensure that only tubulin
heterodimer-associated [
-32P]GTP/GDP was monitored,
the experiment was repeated under conditions in which complexes of
sRB3·tubulin heterodimers were isolated at each time point by passage
over a desalting column prior to analysis. Using this stringent
protocol, it is clear that hydrolysis approaches 50% both in the
presence and absence of excess cold GTP (Fig. 4B), which
indicates that GTP is only hydrolyzed at one of the two E-sites within
the ternary complex. Moreover, this experiment allows an additional
conclusion, namely that GTP exchange is efficiently blocked at both
E-sites of the ternary tandem tubulin heterodimer complex, even at
37 °C. Thus, preferential exchange with cold GTP at either of the
two E-sites can be predicted to shift the observed plateau from 50%
[
-32P]GTP, which is not observed.

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Fig. 4.
GTP is only hydrolyzed at a single E-site in
the ternary tandem tubulin heterodimer complex. A,
[ -32P]GTP was pre-bound to tubulin heterodimers in a
1:1 molar ratio and subsequently mixed with near-saturating
concentrations of sRB3. The hydrolysis state of
[ -32P]GTP, after incubation for the indicated time in
the presence or absence of 1 mM cold GTP, is shown.
B, the hydrolysis state of tubulin heterodimer-associated
[ -32P]GTP, after incubation for the indicated time in
the presence or absence of 1 mM cold GTP, was determined by
analysis of a high molecular fraction (>30 kDa) isolated by separation
on a spin column. Buffer and assay conditions for panels A
and B were the same as in Fig. 2.
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Mutations within Repeat 1 of Op18 Partially Relieve Restrained
Tubulin GTP Hydrolysis within the Complex--
The extended helical
region of Op18/stathmin family members is amphipathic, with conserved
clusters of 3-4 hydrophobic residues phased to the same side of the
helix (23). There are three of these hydrophobic "patches" located
in repeat 1, as depicted in Fig.
5A. These patches are well
conserved among all Op18/stathmin family members, with few conservative
substitutions. To determine whether hydrophobic patches 1-3 are
important for specific tubulin heterodimer-directed activities, mutants
with Ala substitutions of hydrophobic residues were prepared
(designated Op18-pmut1-3; see Fig. 5A). By employing
surface plasmon resonance, the effects of these mutations on the
affinity for tubulin heterodimers was determined in PEM buffer in the
absence of glycerol (Fig. 5B). The data show that mutations
in patch 2 alone were without detectable effect, whereas mutations in
patch 1 or 3 result in significantly decreased affinity. Moreover,
combined mutations in patches 2 and 3 result in a substantial reduction
in the affinity for tubulin heterodimers over and above that of
mutations in patch 3 alone.

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Fig. 5.
Mutational analysis of hydrophobic patches of
the amphipathic helical region of Op18. A, schematic
representation of the conserved hydrophobic patches at repeats 1 and 2 of Op18. The residues shown for each of the patches correspond to the
following positions within the Op18 sequence: patch 1, Leu-47 to
Leu-54; patch 2, Val-68 to Leu-72; and patch 3, Val-82 to Ile-87. The
hydrophobic residues substituted with Ala residues in Op18-pmut1-3
derivatives are underlined. The tandemly arranged
/ -tubulin heterodimers are represented by spheres with
indents indicating the E-site of -tubulin and protrusions indicating
the catalytic loop of -tubulin. B, plasmon resonance
competition experiments at 25 °C using PEM buffer, pH 6.8, in the
absence of glycerol. Graded concentrations of the Op18-pmut derivative
indicated were mixed with 4 µM tubulin heterodimers. The
dotted line depicts complete complex formation at a 1:2
molar ratio of Op18·tubulin heterodimers. C, exchange
rates of the Op18 derivatives indicated were determined as in Fig. 1
but with PEM buffer, pH 6.5, containing 25% glycerol. D,
the initial rates of single-turnover GTP hydrolysis in the presence of
the Op18 derivatives indicated were evaluated in PEM buffer, pH 6.5, containing 17% glycerol.
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The binding affinity of Op18 for tubulin heterodimers is strongly
enhanced at pH values below the physiological range and shows an
optimum at pH 6.5 (13). To facilitate saturated tubulin binding of
Op18-pmut derivatives with decreased binding affinity, tubulin GTPase
and GTP exchange assays were performed at pH 6.5 and in the presence of
25% glycerol. Analysis of GTP exchange activity, presented in Fig.
5C as the half-life of bound [
-32P]GTP,
reveals various levels of decreased GTP exchange inhibitory activity.
The observed differences could not be attributed to various degrees of
saturation of tubulin binding, because each derivative was used at a
sufficient concentration to reach a plateau level in GTP exchange
inhibition (data not shown). Comparison with estimated binding
affinities (Fig. 5B) shows an approximate correlation, which
is consistent with the idea that the stability of the ternary complex
is an important determinant of the magnitude of tubulin GTP exchange
inhibition. Importantly, analysis of the initial rates of tubulin GTP
hydrolysis stimulated by these mutants reveals that substitutions
within patch 2 and patch 3 result in an increased hydrolysis rate,
whereas mutations within patch 1 were without effect (Fig.
5D). It is also noteworthy that combined Ala substitutions
at patches 2 and 3 (i.e. Op18-pmut2/3) have an additive
effect on the rate of hydrolysis. Thus, substitutions in these patches
cause an increase in the conserved GTP hydrolysis rate within the
tubulin heterodimer complex, and each one of the Op18-pmut derivatives
shows a unique pattern of sub-phenotypes as regards the level of
tubulin heterodimer-directed activities.
Patches 2 and 3 of Op18 are both in contact with the
-tubulin
subunit containing the interfaced E-site of the two head-to-tail aligned tubulin heterodimers (see Fig. 5) (4, 8). Because mutations at
these specific hydrophobic patches alter Op18-mediated stimulation of
tubulin GTPase activity, the data are consistent with the idea that GTP
hydrolysis occurs at the interfaced E-site as a result of interaction
with the catalytic loop located on the neighboring
-tubulin subunit,
as originally suggested by the structure of the complex (3). Given that
the rate of GTP hydrolysis is increased after substitutions of
hydrophobic residues at patches 2 and 3, it appears that these patches
are important in keeping the two heterodimers in a configuration that
restrains the otherwise potent GTPase productive interactions that are
facilitated by the head-to-tail arrangement of the heterodimers in protofilaments.
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DISCUSSION |
Op18, RB3, and SCG10 are highly homologous proteins that generate
similar ternary tandem tubulin heterodimer complexes that differ
greatly in their stability (20). Because excess cold GTP shows no
effect on the rate or magnitude of [
-32P]GTP
hydrolysis within the extraordinarily stable RB3·tubulin heterodimer
complex (Fig. 4B), the present analysis indicates that
exchange inhibition is essentially complete at both of the two E-sites
within the complex. One of these E-sites is interfaced between the two
head-to-tail-aligned tubulin heterodimers, and it can be envisioned
that GTP at this interfaced E-site is well enclosed and protected from
exchange (see model in Fig. 5A). However, the other E-site
is completely exposed, and it follows that tubulin GTP exchange
inhibition at this E-site must be explained by an allosteric effect.
It is evident from the present analysis that differences in the
stability of ternary tandem tubulin heterodimer complexes are also
manifested as major differences in the magnitude of GTP exchange
inhibition at 37 °C (Fig. 2, C and D).
Moreover, analysis of GTP exchange inhibition by Op18 derivatives with
destabilizing Ala substitutions in hydrophobic patches also suggests
that the stability of the ternary tandem tubulin heterodimer complex is of primary importance for the potency of GTP exchange inhibition (Fig.
5C). These findings suggest that complex dissociation is required for GTP exchange to occur. We have previously reported that
GTP exchange inhibition by Op18 at 37 °C is partially dependent on
the non-helical N-terminal region (5), and the present studies extend
this finding to the sRB3 and sSCG10 proteins (Fig. 2, C and
D). Binding analysis shows that the non-helical N-terminal region of Op18 contributes to the affinity of tubulin heterodimer binding (Fig. 3A), and it follows that the observed
importance of this region for GTP exchange inhibition can readily be
explained as an indirect consequence of increased complex stability.
Consistent with this interpretation, it is clear from analysis of GTP
exchange inhibition at 0 °C that the extended helix of Op18 is
sufficient for optimal inhibition of GTP exchange at both sites of the
complex (Fig. 3B).
The present determination of the stoichiometry of GTP hydrolysis within
the ternary tandem tubulin heterodimer complex, combined with the
phenotype of Ala substitutions at distinct hydrophobic clusters along
the face of the extended helix of Op18, is most consistent with GTP
hydrolysis occurring at the interfaced E-site only (see Fig.
5A). The most likely mechanism involves interactions between
the interfaced E-site and the catalytic loop located on the neighboring
-tubulin subunit, as originally suggested by the head-to-tail
arrangement of heterodimers in the complex (3). This simple model is
also consistent with the finding that the rate of tubulin GTP
hydrolysis within the complex is independent of the non-helical
N-terminal region of Op18/stathmin family members (Fig. 2). It is
notable that the conserved rate of GTP hydrolysis within a ternary
tandem tubulin heterodimer complex is several orders of magnitude lower
than during polymerization-facilitated interactions between the E-site
and the catalytic loop of an adjacent heterodimer. This indicates that
Op18/stathmin family members keep the two heterodimers in a
configuration that restrains the otherwise potent GTPase productive
interactions facilitated by the head-to-head alignment of heterodimers
in protofilaments (10). In the present study, we have identified
structural hydrophobic motifs that are at least in part responsible for
restraining GTPase productive interactions, as evidenced by the
observed phenotypes of Ala substitutions in the conserved hydrophobic
patches 2 and 3 of Op18 (Fig. 5). It is of particular interest that
substitutions at patch 2 resulted in increased GTP hydrolysis within
the ternary tandem tubulin heterodimer complex, with minimal effects on
tubulin heterodimer binding affinity and GTP exchange inhibition. This indicates the specificity and importance of these hydrophobic patches
for restraining GTPase productive interactions between the two
head-to-tail-aligned tubulin heterodimers within the ternary complex.
The present mutational analysis of conserved hydrophobic patches
reveals a unique pattern of sub-phenotypes regarding the level of
tubulin heterodimer binding, GTP exchange, and hydrolysis. Although
there is a correlation between tubulin heterodimer binding affinity and
GTP exchange inhibition, our data uncouple tubulin heterodimer binding
affinities from the stimulation of GTP hydrolysis. However, in order to
generate GTPase productive ternary complexes, it is essential that
binding should occur with sufficient positive cooperativity such that
the formation of hydrolysis-incompetent binary complexes containing a
single tubulin heterodimer would be minimized. In the absence of
glycerol, the Op18-pmut1 derivative shows a positive binding
cooperativity that is too low to efficiently form GTPase productive
tandem tubulin heterodimer complexes (data not shown). This is also
manifested as a low tubulin heterodimer binding affinity in the present
plasmon resonance competition analysis, which was performed in a
glycerol-free buffer (Fig. 5B). Consistent with decreased
positive binding cooperativity under these conditions, Op18-pmut1 is
inefficient in stimulating tubulin GTP hydrolysis if the assay is
performed in the absence of glycerol (19). However, the addition of
glycerol to the buffer increases positive binding cooperativity (data
not shown), and allows efficient generation of ternary tandem tubulin
heterodimer complexes with normal levels of GTP hydrolysis (Fig.
5D). This illustrates that glycerol, by altering cooperative
tubulin heterodimer binding properties, may also alter specific
phenotypes of mutant derivatives, which may in some cases confuse
interpretation of in vitro results. In the present study,
glycerol was included in the different assays to allow comparison of
truncated/mutated derivatives with decreased tubulin heterodimer
binding affinity, generating conditions in which binding approached
saturation in all cases. It should be noted, however, that the
Op18-pmut2 and 3 derivatives showed increased tubulin GTPase
stimulatory activities both in the absence and presence of glycerol
(data not show). Moreover, although decreased binding cooperativity may
explain an apparent decrease in the stimulation of tubulin GTP
hydrolysis as outlined above, the observed increase in stimulation of
GTP hydrolysis caused by mutations of Op18 patches 2 and 3 cannot be
explained by alterations in the level of binding cooperativity.
That Op18 modulates the GTP exchange and hydrolysis properties of
tubulin heterodimers has evoked speculation previously on the potential
significance of these activities for the mechanism underlying
catastrophe promotion (5). However, catastrophe promotion, which
requires the non-helical N-terminal region of Op18 (14, 24), does not
depend on the formation of GTPase productive/GTP exchange-inhibited
ternary complexes. Hence, the present analysis, which was prompted by
recent structural insights into the architecture of the ternary complex
(3, 4), indicates a conserved and very different role for these GTP
exchange/hydrolysis modulatory activities, namely to prevent futile
cycles of GTP hydrolysis.