(Received for publication, August 3, 1994)
From the
Microtubule-associated protein (MAP)-2 is a multidomain
cytoskeletal protein that copurifies with brain microtubules (MTs)
through repeated cycles of warm polymerization and cold disassembly.
Recent equilibrium binding studies of high molecular weight MAP-2ab to
taxol-stabilized MTs suggest that the interactions are highly
cooperative, as indicated by sigmoidal binding curves, non-linear
Scatchard plots, and an apparent all-or-none response in MAP binding in
titration experiments (Wallis, K. T., Azhar, S., Rho, M. B., Lewis, S.
A., Cowan, N. J., and Murphy, D. B.(1993) J. Biol. Chem. 268,
15158-15167). To learn more about the mechanism of MAP-2 binding
to MTs, we investigated the binding properties of bacterially expressed
MT-binding region (MTBR) of bovine brain MAP-2. Scatchard plots of the
binding data showed no evidence of cooperativity, as reflected by the
linear plots of t;ex2html_html_special_mark_amp;ngr;/[MTBR] versus t;ex2html_html_special_mark_amp;ngr;. The stoichiometry was
1-1.1 mol of MTBR/mol of tubulin dimer, and the dissociation
constant for the MTBR was 1.1 µM. Bovine brain tau protein
competitively inhibited MAP-2 binding, as evidenced by an increased K
value for MTBR binding to MTs. Although
the second repeat peptide m
(VTSKCGSLKNIRHRPGGG) is thought
to play a dominant role in MAP-2 binding to MTs, a MTBR mutant (with
m
replaced by the third octadecapeptide repeat
m
) displays an K
of 2.8
± 0.1 µM and stoichiometry of 0.9 ± 0.05 mol
of MTBR/mol of tubulin dimer. Another mutant with additional copies of
the second repeat, designated by us as
MTBR[m
m
m
], displayed
noncooperative binding with a K
of 0.53
± 0.05 µM and a stoichiometry of 2.2 ± 0.2
mol of mutant MTBR/tubulin dimer. Equilibrium sedimentation experiments
demonstrated that the wild-type MTBR is monomeric, whereas
MTBR[m
m
m
]
selfassociates to a stable dimer over the concentration range used in
our MT binding studies. This finding indicates that only one of the two
MT-binding sites on the dimer is probably linked to a microtubule at
any given time.
The microtubule (MT) ()cytoskeleton achieves greater
stability and rigidity through cross-linking by microtubule-associated
proteins, or MAPs (Wuerker and Palay, 1969; Ellisman and Porter, 1980;
Okabe and Hirokawa, 1988; Olmsted, 1986). Among these cross-linkers is
the brain-specific protein MAP-2, which appears in embryonic and
neonatal brain in a low-molecular weight form known as MAP-2c and in
adult brain as the high molecular weight form MAP-2a (Wiche, 1989;
Matus, 1990; Tucker, 1990). Starting from the amino terminus, the
multifunctional domains of MAP-2 include: a high affinity binding site
for the regulatory subunit of protein kinase A (Obar et al.,
1989; Rubino et al., 1989), an extended projection-arm
containing over 1300 amino acid residues in the adult form, a hinge
region possessing several protease-accessible cleavage sites (Vallee
and Borisy, 1977; Joly et al., 1989), and the
microtubule-binding region (MTBR) comprising a non-identical peptide
repeat triad (Lewis and Cowan, 1988; Lewis et al., 1989).
Thombin cleavage of bovine brain MAP-2ab occurs at a position
corresponding to residue 1628 in the mouse MAP-2 sequence (Joly et
al., 1989) yielding two fragments; the smaller 201-amino acid
COOH-terminal portion promotes MT self-assembly as effectively as
intact MAP-2 (Flynn et al., 1987; Joly et al., 1989).
Synthetic peptides corresponding to the second sequence repeat within
the MT-binding region can promote tubulin polymerization (Joly et
al., 1989), and these peptides displace intact MAP-2ab from
assembled microtubules (Joly and Purich, 1990); however, such peptides
display significantly lower affinity (K
0.3-0.5 mM) than does the entire
MT-binding region (K
1-3
µM). In recent studies of the equilibrium binding of high
molecular weight MAP-2ab to taxol-stabilized microtubules, Wallis et al.(1993) observed highly cooperative binding, as indicated
by sigmoidal binding curves, non-linear Scatchard plots, and an
apparent all-or-none response in MAP binding in titration experiments.
These investigators also directly observed clusters of MAP-2 molecules
on microtubules using immunoelectron microscopy, a finding that is
consistent with cooperative binding of MAP-2 to the tubule lattice.
Their other studies with truncated versions of mouse MAP-2 also
suggested that the region conferring cooperativity may be located in or
near the MT-binding site located near the carboxyl terminus. As part of
our analysis of the molecular recognition properties of MAP-microtubule
interactions, we have used bacterially expressed
[
H]leucine-labeled, MT-binding region of bovine
MAP-2 to examine the binding properties of this region in the absence
of other domains found in MAP-2. We now report our findings indicating
the complete absence of cooperativity in the binding of the MTBR to
taxol-stabilized microtubules. We also discuss results of related
binding experiments with mutant forms of the tubule-binding region
containing (a) either first and third non-identical repeats
modified to resemble the second sequence repeat or (b) an
extra copy of the third repeat in place of the second repeat.
To determine whether the multidomain nature of MAP-2 regions
contributes to the cooperativity in MAP-2 binding to MTs observed by
Wallis et al.(1993), we chose to restrict our investigation to
the equilibrium binding properties of MAP-2's MT-binding region.
We used two different bovine MAP-2 fragments, one (designated
MTBR) corresponding to positions 1509-1828 in the
mouse sequence, and the second (MTBR
) spanning the
sequence from the thrombin-cleavage site following position 1628 to the
carboxyl terminus (see diagram in Fig. 1). Quantitative analysis
of MAP binding to microtubules was facilitated by incorporation of
[
H]leucine into the MTBR during bacterial
expression. We subsequently established the specific radioactivity by
using protein determinations based on amino acid analysis of MTBR, with
norleucine added prior to acid hydrolysis to serve as an internal
reference standard. After incubating the MAP with MTs, we used
ultracentrifugation to fractionate microtubule-bound and free MTBR by
recovering the pellet and supernatant fractions, respectively. The
binding data from experiments with MTBR
and
MTBR
yielded linear Scatchard plots (Fig. 2, A and B). We carried out these experiments over a
reasonably wide range of saturation, corresponding in these particular
experiments to fractional occupancies in the ranges of 0.08-0.9
and 0.06-0.75 for MTBR
and MTBR
,
respectively. Such linearity was observed with both MTBR constructs,
although each displayed slightly different affinity and stoichiometry
of binding, based on their slope and horizontal intercept values. The
linear nature of the Scatchard plots has been observed in all of our
binding experiments, and there is no evidence of cooperativity. It
should be emphasized that our solution conditions were identical to
those routinely employed in our laboratory for analyzing microtubule
self-assembly (i.e. 0.1 M zwitterionic buffer (in
this case, MES) at pH 6.8 with magnesium ion, GTP, and EGTA). Our
conditions are in fact quite similar to those applied by Wallis et
al.(1993), except that we assembled microtubules prior to addition
of taxol to avoid aberrant polymeric forms of tubulin, and we did not
employ non-ionic detergent or chemically iodinated MAP-2.
Figure 1:
Schematic diagram of MAP-2 and four
microtubule-binding region fragments. The multiple domain nature of
MAP-2ab is illustrated by the presence of a protein kinase R site (cross-hatched), an extended projection-arm domain,
and a microtubule-binding region containing three non-identical
octadecapeptide repeats, m
, m
, and m
(represented as shaded boxes). Also illustrated are
truncated bovine MAP-2 MT-binding regions: MTBR
, a
fragment spanning from just prior to the MAP-2c splice junction site at
position 1519 to the COOH terminus; MTBR
, a smaller
product from the thrombin cleavage site through the COOH terminus;
MTBR
[m
m
m
]
and
MTBR
[m
m
m
],
mutant molecules with the second non-identical repeat replaced with a
copy of the third repeat and with the first and third repeats changed
to more closely resemble the second repeat,
respectively.
Figure 2:
Scatchard binding plots for MAP-2
microtubule-binding fragment interactions with taxol-stabilized
microtubules in a cosedimentation assay. A, MTBR displayed a dissociation constant of 1.2 ± 0.25
µM. B, MTBR
had a K
= 1.8 ± 0.1
µM. Both fragments showed a maximum stoichiometry of
approximately 1 mol/mol of tubulin dimer. In these experiments, the
tubulin concentration was held at 2.75 µM while the MTBR
concentration was varied from 0.3 to 9
µM.
The
stoichiometry of binding was found to correspond to about 1.1 ±
0.1 and 1.0 ± 0.1 mol of MTBR and
MTBR
/mol of tubulin dimer. We obtained dissociation
values of 1.2 ± 0.25 and 1.8 ± 0.1 µM,
respectively, with these proteins. As an additional check of our
stoichiometry and as a means for analyzing competition between intact
tau protein with MAP-2 MTBR, we carried out the binding experiment
shown in Fig. 3. In this case, the concentration of
radioactively labeled MT-binding region was varied from 0.3 to 9
µM in the absence and from 1.2 to 7 µM in the
presence of a fixed concentration (2 µM) of bovine brain
tau protein. The observed equilibrium binding behavior is consistent
with direct competition of tau and MAP-2 MTBR, based on the fact that
the linear plots converge at a common horizontal intercept
corresponding to a stoichiometry of 1.05 ± 0.1 mol/mol. The
apparent affinity, reflected by the change in the slope, was reduced in
the presence of tau protein, and we calculated a dissociation constant
(K
) of 5.2 µM for tau binding by using the
following equation for competitive ligand
binding.
Figure 3:
Binding of MTBR to
taxol-stabilized MTs in the presence of 2 µM tau protein.
Tau appears to compete with the microtubule binding region of MAP-2 for
the same site on tubulin; the apparent affinity was lower in the
presence of tau as evidenced by the change in slope, but the
stoichiometry remained unchanged.
That the Scatchard plot in the presence of tau protein was not biphasic again suggests that intact tau protein binds with a similar stoichiometry and without any evident cooperativity.
Previous
experimental evidence indicated that the second repeated peptide
m (VTSKCGSLKNIRHRPGGG) is likely to play a dominant role in
MAP-2 binding to microtubules (Joly et al., 1989; Joly and
Purich, 1990), and the first and third peptide repeats, designated
m
(VKSKIGSTDNIKYQPKGG) and m
(AQAKVGSLDNAHHVPGGG), were completely ineffective in promoting
tubulin polymerization. Recent studies of tau protein by Goode and
Feinstein (1994), however, indicate that the so-called inter-repeat
region separating the corresponding first and second repeat sequences
of tau also plays a role in the binding of tau protein to microtubules.
Through site-directed-mutagenesis, we completely eliminated the m
sequence and replaced it with an identical length copy of the
third octadecapeptide repeat m
. We reasoned that such a
substitution should completely block MAP-2 MTBR binding to microtubules
if the second repeat is the only participant in microtubule binding. As
shown in Fig. 4, the mutant form
MTBR
[m
m
m
]
still displayed affinity, albeit somewhat reduced, for microtubules.
The apparent dissociation constant was determined to be 2.8 ±
0.1 µM, and an extrapolated value of 0.9 ± 0.05 was
obtained for the number of MTBR molecules bound to each tubulin dimer.
This reduction in binding affinity significantly limits the ability of
the mutant MTBR to stimulate the polymerization of tubulin (see Fig. 4, inset).
Figure 4:
Scatchard plot for the MT binding of MTBR[m
m
m
]
mutant lacking the second repeat sequence. A dissociation constant
value of 2.8 ± 0.1 µM was obtained, compared to 1.2
± 0.25 µM for the wild-type fragment,
MTBR
. This mutant was inefficient in promoting MT
assembly compared to the wild-type fragment as monitored by light
scattering with 0.73 µM MTBR and 11 µM tubulin (see inset).
We were also interested to learn
whether conversion of the first and third repeats to sequences
resembling the second repeat would improve the binding affinity of our
recombinant MAP fragment. We designed mutagenesis experiments to
evaluate the relative importance of various amino acid side chains
within the second repeat m, and this permitted us to
identify critical residues involved in MT binding. (
)We then
altered those corresponding positions in the first and third repeats
(indicated by boldface type) in order to bring the first and third
sequences into compliance with what dominant interactions in the
m
sequence. These forms are termed m
for
VKSKIGSLKNIRHRPGGG and m
for
AQAKVGSLKNARHRPGGG. Chemically synthesized peptide
corresponding to m
and m
display the same
potency as synthetic m
in displacing MAP-2
microtubule-binding region from assembled MTs (Coffey et al.,
l994). To assess the effect of substituting these sequences into the
MAP-2 MTBR, we constructed and expressed the mutant
MTBR
[m
m
m
],
spanning the region from position-1629 (thrombin site) to position 1828
(COOH terminus). The data presented in Fig. 5demonstrate that
this novel MAP-2 MT-binding region mutant displays linear equilibrium
binding behavior, and the affinity of the mutant for microtubules was
increased somewhat, as indicated by the dissociation constant (0.53
± 0.05 µM). The observed stoichiometry of binding
was increased to 2.2 ± 0.2 mol of mutant MTBR/tubulin dimer. It
should be noted that bacterially expressed MTBR
shows no
evidence of any covalently cross-linked MTBR oligomers using
time-of-flight mass spectrometry. (
)To learn more about the
self-association of MTBR
and
MTBR
[m
m
m
]
in solution, we analyzed the sedimentation equilibrium behavior of MTBR
using the Beckman XLA ultracentrifuge to learn whether the monomer
displays evidence of self-association. Even at concentrations of
0.6-0.7 mg/ml, which are far greater than those employed in our
equilibrium binding experiments, we found that greater than
90-95% of the MTBR
was monomeric (Fig. 6).
This can be shown for the upper curve, which displays a close
correspondence of the theory line (based on a monomer molecular weight
of 22,000) with the experimental data points in plots of protein
concentration versus radius. When companion experiments were
conducted with the
MTBR
[m
m
m
]
mutant, the lower curve was obtained. We observed a very good fit for a
species of 37,000 molecular weight, and the theory line (dashed) for a monomer clearly demonstrates the
incompatibility of a monomeric species with this sedimentation
behavior. Taking this together with the binding data presented in Fig. 5, we are drawn to the conclusion that the
MTBR
[m
m
m
] mutant binds as a dimer, most likely to a
single site on assembled microtubules.
Figure 5:
Scatchard plots for binding of MTBR and a MTBR
[m
m
m
]
mutant containing additional copies of m
-like sequences in
place of m
and m
. Wild-type MTBR again
displayed unit stoichiometry, but we obtained a stoichiometry of
approximately 2.2 mol/mol of tubulin for the latter. This mutant also
displayed slightly higher affinity (K
= 0.53 ± 0.05 µM) compared to
wild-type (1.2 ± 0.25
µM).
Figure 6:
Sedimentation equilibrium behavior of the
mutant MTBR[m
m
m
]
and wild-type MTBR
. The
theoretical gradient for the known molecular weight of 22,000 for
MTBR
is shown as a solid line superimposed on
the experimental values.
MTBR
[m
m
m
]
behaved as a much larger species shown with a best fit of M
37,000 (dashed line), suggesting that
upward of 90% of the mutant was dimeric.
MAP-2 is thought to play important roles in cross-linking cytoskeletal elements in neurons, and this structural MAP may be responsible for cross-bridges between microtubules as well as between neurofilaments and microtubules. Indeed, MAP-2 can cross-link microtubules and neurofilaments in vitro (Aamodt and Williams, 1984; Flynn and Purich, 1987), and the microtubule-binding region derived from thrombin cleavage of MAP-2ab can bind to both microtubules and neurofilament light chain (Flynn et al., 1987). Kowalski and Williams(1993) also used video microscopy with differential interference contrast optics to demonstrate that MAP-2 binding can greatly suppress dynamic instability of MTs. Likewise, Yamauchi et al.(1993) employed length redistribution measurements to show that microtubule dynamics are suppressed by MAP-2 and tau, and even synthetic octadecapeptide analogues corresponding to the second repeat in the MAP-2 MT-binding domain can stabilize MTs against dynamic instability. Direct stabilization of tubulin in its polymerized conformation does not require cross-linking, indicating the stabilizing effects of MAP-2 on MTs need not require any cooperative binding interactions with microtubules through a contiguous set of peptide repeats.
Despite conflicting observations that MAP-2 can bind
irreversibly to microtubules (Job et al., 1985) and that
fluorescently labeled MAP-2 was readily exchangeable (Scherson et
al., 1984; Olmsted, 1986; Flynn, 1988), no detailed analysis of
equilibrium binding of MAP-2 to microtubules appeared prior to that
presented by Wallis et al.(1993). The experimental findings
presented here indicate that interactions of the MAP-2 MT-binding
region with microtubules can be characterized as independent and
non-cooperative. Furthermore, the stoichiometry of MTBR binding to
microtubules suggests that there is little or no interference of MAP
binding by other bound MAP molecules, even when a stoichiometry of 1
mol/mol is achieved. While the basis for the complexity of MAP-2
binding observed by Wallis et al.(1993) remains to be
elucidated, some mention should be made concerning the non-ideality of
MAP-2 in solution, especially in its high molecular weight form as
isolated by recycling brain microtubule fractions using warmth-induced
assembly and cold depolymerization protocols. It has been our
experience that MAP-2 readily undergoes aggregation when separated from
microtubules, and this is likely to be the result of ionic interactions
between the highly acidic projection-arm region (isoelectric point
4.7-4.9) and the highly basic microtubule-binding region
(isoelectric point 10.2-10.5), as determined by non-equilibrium
gel electrophoresis (Flynn et al., 1987). Interestingly,
Wallis et al.(1993) prepared an apparently monodisperse
solution of MAPs by heat incubation with 0.1% Triton X-100 at 37 °C
for 10 min, and they found that 99% of the MAP-2 fraction remained
soluble after ultracentrifugation. While the presence of the nonionic
detergent did not appear to alter the salt-sensitive binding of MAPs to
taxol-stabilized microtubules, these observations do not prove that
such MAP-2 preparations are truly monodisperse in a thermodynamic
sense. Furthermore, Wallis et al.(1993) introduced I into MAP-2 by iodination in the presence of the oxidant
chloramine-T (Hunter and Greenwood, 1962), and the influence of such
treatment on MAP-2 binding properties remains to be assessed. It is
noteworthy, however, that Joly and Purich(1990) employed trace-labeled
[
P]MAP-2 in binding experiments with
taxol-stabilized microtubules, and they observed hyperbolic binding
behavior in the absence or presence of the peptide analogue
corresponding to the second non-identical repeat in MAP-2. Our
metabolic labeling of the MTBR with [
H]leucine
during bacterial expression completely eliminates any complications
arising from chemical iodination or enzymatic phosphorylation. We
should also note that Kowalski and Williams(1993) observed no evidence
of cooperativity in their studies of microtubule assembly/disassembly
dynamics as a function of MAP-2 concentration.
Despite our findings of non-cooperative MAP-2 MTBR binding to taxol-stabilized microtubules, we do not dispute the possible occurrence or potential significance of cooperative interactions resulting from the presence of projection arms in intact high molecular weight MAP-2ab. Indeed, Wallis et al.(1993) adduced electron microscopic evidence for the clustering of MAP-2ab on microtubules, and they observed that some regions of the tubule lattice were densely labeled with MAP-2ab while other regions along the lattice were only sparsely decorated with this MAP. Such binding behavior is reminiscent of the cooperative binding behavior of Chlamydomonas flagellar dynein to singlet brain tubules (Haimo et al., 1979). The latter investigators found that binding of the first dynein molecule favored the formation of clusters in what appeared to be a zipper-like cooperative process.
The occurrence of
multiple non-identical sequence repeats in the microtubule-binding
regions of tau, MAP-2, and MAP-4 has invited speculation that these
proteins bind to MTs by engaging several or all of the repeats in the
binding interaction. Lewis et al. (1989) first considered the
possibility that the inter-repeats might be of sufficient length to
permit the repeats to bind to neighboring subunits in the microtubule
lattice. Such an attractive proposal could explain observations that
high molecular weight MAP-2ab binds to MTs with a stoichiometry
corresponding to 1 MAP/3-4 tubulin molecules (Joly and Purich,
1990), although the presence of the long projection-arm domains may
also explain this stoichiometry on the basis of steric hindrance. The
stabilizing effects of MAP-2 in promoting MT self-assembly and in
suppressing MT assembly/disassembly dynamics have also been interpreted
as indicating the likelihood of multiple interacting sites for binding
several repeats simultaneously. Nonetheless, our finding that the MAP-2
MTBR binds with unit stoichiometry relative to tubulin dimers precludes
such models, especially when one considers that the affinity of MAP-2
MTBR remains unchanged as a function of binding site occupancy (hence
the linear Scatchard plots). Furthermore, earlier studies demonstrated
that the second repeat peptide can promote tubulin polymerization (Joly et al., 1989), and such 18- and 21-amino acid peptide
analogues can also markedly suppress MT dynamic instability (Yamauchi et al., 1992). Why then does MAP-2 have such multiple repeats?
One idea is that the presence of these repeats increases the frequency
with which the initial, albeit weak, complexation reaction of MAP and
microtubule proceeds. This would effectively increase the bimolecular
rate constant for MAP-2 addition to its microtubule binding site, and
such initial complexes might then isomerize to form tighter complexes
preferentially involving the second repeat. Another possibility is that
one or more of the inter-repeats are involved in MAP binding, and
several reports on the binding interactions of tau protein are in
harmony with this proposal (Butner and Kirschner, 1991; Goode and
Feinstein, 1994). The latter investigators used deletion mutants to
demonstrate that the first inter-repeat contributed markedly to tau
protein binding to microtubules, and they also showed that synthetic
peptides corresponding to the the first inter-repeat can promote
tubulin polymerization. Ainsztein and Purich(1994) recently observed
that protein kinase C phosphorylation of Ser-1703, Ser-1711, and
Ser-1728 in the bovine brain MAP-2 MTBR were all required to eliminate
the ability of the MTBR to stimulate tubulin polymerization. Ser-1703
lies within the first inter-repeat, Ser-1711 is located in the second
non-identical repeat motif, and Ser-1728 is situated in the second
inter-repeat. Thus, it would appear that the non-identical repeats lie
in tandem such that multiple contacts are made with a tubulin in the MT
lattice; this could occur while still permitting close packing of MTBR
molecules to the extent of 1/tubulin dimer. This proposal would also
explain the lower affinity of small peptide analogues that promote
tubulin polymerization, especially when one recalls that binding
affinity is exponentially related to the sum of individual binding
energies for sub-site interactions of ligands with protein binding
sites. The fact that the MTBR [m
m
m
] mutant binds,
albeit more weakly, even in the absence of a second repeat sequence,
argues that other regions in the MTBR are contributing to the binding
energy. Nonetheless, we should stress that the MTBR
[m
m
m
] mutant does
not promote tubulin polymerization to any significant level relative to
wild-type MTBR.
We recently demonstrated that additional copies of
the second repeat do not greatly improve the binding affinity of
recombinant MTBR to taxol-stabilized microtubules (Coffey et
al., 1994). In those experiments, changes in binding affinity were
evaluated in terms of the concentration of an m peptide
analogue needed to displace the mutant MTBR from assembled tubules. We
also found that introduction of m
or m
individually or in combination exhibited only slightly higher
affinity for microtubules. While the findings presented here add
evidence for the proposal that the second repeat plays a dominant role
in MAP-2 binding to MTs, we have not as yet systematically investigated
the role of the inter-repeat regions. Moreover, future studies will be
needed to understand the sedimentation equilibrium behavior of mutant
and wild-type forms in order to determine the protein concentration
dependence of oligomerization reactions.
We found that tau protein was an effective competitive inhibitor of MAP-2 MTBR binding to microtubules, and our experimental results agree with published reports indicating the lack of cooperativity in tau binding (Butner and Kirschner, 1991; Gustke et al., 1992; Biernat et al., 1993; Goode and Feinstein, 1994). Interestingly, two of the three tau repeat peptides can promote MT assembly (Ennulat et al., 1989), whereas only the second peptide repeat in MAP-2 displays this property. Titration experiments of tau peptide binding to microtubules indicate that 2 mol of peptide bind/mol of tubulin (Melki et al., 1991), and one might have anticipated the possibility for high affinity, cooperative binding by this microtubule-associated protein. All published experiments suggest that tau protein has about the same affinity for taxol stabilized microtubules as that observed with MAP-2 MT binding domain.