©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Non-cooperative Binding of the MAP-2 Microtubule-binding Region to Microtubules (*)

(Received for publication, August 3, 1994)

Richard L. Coffey Daniel L. Purich (§)

From the Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Health Science Center, Gainesville, Florida 32610-0245

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(2) (VTSKCGSLKNIRHRPGGG) is thought to play a dominant role in MAP-2 binding to MTs, a MTBR mutant (with m(2) replaced by the third octadecapeptide repeat m(3)) 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[mm(2)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[mm(2)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.


INTRODUCTION

The microtubule (MT) (^1)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 approx 0.3-0.5 mM) than does the entire MT-binding region (K approx 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 [^3H]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.


EXPERIMENTAL PROCEDURES

Materials

Isopropyl-beta-D-thiogalactopyranoside, MES, EGTA, MgCl(2), KCl, dithiothreitol, phenylmethylsulfonyl fluoride (PMSF), DNase, ampicillin, Tris-HCl, sodium dodecyl sulfate, and GTP were purchased from Sigma. Phosphocellulose-P11 was a Whatman product. Taxol was obtained from Dr. Matthew Suffness of the National Cancer Institute, Bethesda, MD. [^3H]Leucine was from Amersham Corp., and [-P]ATP was from ICN. The Amplitaq cycle sequencing kit was purchased from Perkin-Elmer Corp. New England Biolabs supplied restriction endonucleases, T4 DNA ligase, and T4 polynucleotide kinase. Synthetic oligonucleotides were produced at the DNA Synthesis Core at the University of Florida. Promega supplied Taq DNA polymerase, while deoxynucleoside triphosphates were from Pharmacia Biotech Inc. Glucose, casamino acids, CaCl(2), and NaCl were from Fisher. Chloramphenicol was purchased from Boehringer Mannheim.

Methods

Bacterial Expression and Protein Purification

For details of the cloning and sequencing the bovine MAP-2 MT-binding region as well as vector construction; see Coffey et al.,(1994). Mutants MTBR[mm(2)m] and MTBR[m(1)m(3)m(3)] were created using cassette mutagenesis (Sambrook et al., 1989) and polymerase chain reaction mutagenesis (MacPherson, 1991), respectively, and sequenced to ensure that only the desired mutations were present. Expression plasmids using the T7 gene 10 promoter were transformed into Escherichia coli BL21(DE3) and grown on LB plates containing 50 µg/ml ampicillin and 50 µg/ml chloramphenicol. A single colony was used to inoculate a 5-ml LB culture containing 50 µg/ml chloramphenicol and grown overnight at 37 °C. Two ml of this culture were diluted into 100 ml of medium consisting of 48 mM Na(2)HPO(4), 22 mM K(2)HPO(4), 8.6 mM NaCl, 19 mM NH(4)Cl, 0.05% casamino acids, 0.001% thiamine, 1.0 mM MgCl(2), 0.2% glucose, 50 µg/ml ampicillin, and 50 µg/ml chloramphenicol. This culture was grown at 37 °C to an optical density of 0.3 (600 nm) when 200 µCi of [^3H]leucine were added. Growth at 37 °C was continued for 15 min, isopropyl-beta-D-thiogalactopyranoside was added at a concentration of 1 mM, and growth was continued for another 3 h. Cells were harvested at 1500 times g for 10 min and resuspended in 4 ml of MEM buffer (100 mM MES, 1 mM EGTA, and 1 mM MgCl(2), pH 6.8). They were pelleted again at 1500 times g for 10 min and resuspended in 1 ml of buffer containing 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1.0 mM MgCl(2), 1.0 mM dithiothreitol, 21 units/ml DNase, and 0.5 mM PMSF. Cells were lysed by three rounds of freezing and thawing, using liquid nitrogen and a 37 °C water bath, and centrifuged at 16,000 times g for 20 min. The pellet fraction containing insoluble inclusion bodies was rinsed with MEM buffer and resuspended in 0.5 ml of the same buffer supplemented with 0.75 M NaCl, 1 mM dithiothreitol, and 0.5 mM PMSF. This suspension was then rapidly heated to a final temperature of 80 °C and held for 10 min to solubilize the heat-stable MAP-2 microtubule-binding fragment. Subsequent incubation on ice for 20 min and centrifugation at 16,000 times g for 30 min served to remove most of the aggregated contaminating proteins. The supernatant predominantly contained intact MAP-2 MT-binding fragment, and this was diluted to a final volume of 2.5 ml with H(2)O and loaded on a 0.2-ml phosphocellulose column. The bound MAP-2 MT-binding fragment was washed with MEM buffer and eluted with 1.0 ml of a high ionic strength buffer (100 mM MES, 1.0 mM EGTA, 1.0 mM MgCl(2), 1.0 mM dithiothreitol, 1.0 M NaCl, pH 6.8). The volume of the eluent was reduced to 0.5 ml by vacuum concentration, followed by intensive dialysis in MEM buffer containing 1 mM dithiothreitol. Centrifugation at 100,000 times g for 20 min ensured that the dialysate contained only soluble MAP-2 MT-binding fragment. The solution was aliquoted, frozen in liquid nitrogen, and stored at -70 °C for later use. This method typically yielded 500-1000 µg of MT-binding fragment at a specific activity of 1000-2000 cpm/µg, which was greater than 99% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining or fluorography.

Tubulin Purification and Assembly Assays

Bovine brain microtubule protein and tubulin were further purified by phosphocellulose chomatography as outlined elsewhere (Joly and Purich, 1990). Tubulin polymerization experiments were carried out in MEM buffer (100 mM MES, 1 mM EGTA, 1 mM MgCl(2), pH 6.8) with 1 mM GTP and 1 mM dithiothreitol. Tubulin (1.1 mg/ml) was assembled with 0.73 µM MT-binding domain. The assembly process was monitored using a Cary 210 spectrophotometer at 350 nm in a cuvette thermostated to 37 °C.

Binding Experiments and Scatchard Analysis

Taxol-stabilized microtubules were prepared by assembling pure tubulin at 5 mg/ml in MEM buffer with 1 mM GTP at 37 °C for 15 min. Taxol was then added at a concentration of 50 µM, incubation at 37 °C was continued for 15 min, and the solution was diluted to 2.5 mg/ml tubulin and held at 37 °C. Each binding assay contained 0.25 mg/ml tubulin (taxol-stabilized MTs), 100 mM MES, 1 mM EGTA, 1 mM MgCl(2), 1 mM GTP, 2.5 µM taxol, and 60 mM KCl at pH 6.8 in a final volume of either 50 or 100 µl. [^3H]MAP-2 MTBR was present at various concentrations ranging from 0.3 to 9 µM and was always centrifuged at 100,000 times g for 15 min immediately prior to the experiment to remove any aggregated protein. These components, except tubulin, were mixed and warmed to 37 °C. Then taxol-stabilized microtubules were added and gently mixed by pipetting with a pipette tip cut at the end such that the opening was at least 1 mm in diameter to minimize shearing forces. This solution was incubated at 37 °C for 20 min, transferred to centrifuge tubes precoated with 10 mg/ml BSA, and centrifuged at 100,000 times g for 15 min at 37 °C in a Beckman Airfuge. The supernatant was removed, and the pellet was rinsed with MEM buffer and resuspended in 30 µl of 0.5% SDS. The amount of MTBR bound to microtubules was determined by scintillation counting of the pellet fraction. The data were plotted as a ratio of t;ex2html_html_special_mark_amp;ngr;/[MTBR]versust;ex2html_html_special_mark_amp;ngr;, where t;ex2html_html_special_mark_amp;ngr; is the fractional saturation of sites on assembled microtubules and [MTBR] is the concentration of uncomplexed MTBR. The apparent dissociation constant and limit stoichiometry were calculated from the slope and intercept as determined by linear regression (Scatchard, 1946). Experiments were conducted in duplicate, and the range is indicated by the deviation.

Sedimentation Equilibrium Experiments

To investigate the self-associative nature of wild-type and mutant MAP-2 MTBRs, samples were centrifuged at 16,500 rpm in a Beckman XLA Analytical Ultracentrifuge at 23 °C in 100 mM MES, 1 mM EGTA, 1 mM magnesium chloride (pH 6.8). Protein concentrations were 118 µM MTBR and 112 µM MTBR [mm(2)m]. Ultracentrifugation runs were continued for 72 h for the former and 30 h for the latter, at which times sedimentation equilibrium was attained. The protein concentration gradient across the cell was directly monitored by UV absorbance at 256 nm, and these sedimentation data were subsequently analyzed using MLAB software.

Protein Concentrations

We used the method of Bradford (1976). To standardize this method for tubulin and MAP-2 MTBR, we performed acid hydrolysis and amino acid analysis of these two proteins to determine nanomolar quantities of individual amino acids. Based on the known sequence for the MAP-2 fragment and the averaged abundances of individual amino acids for all of the mammalian tubulins in the PIR data base, the nanomolar amounts of MTBR and tubulin were calculated. Amino acid analysis was performed twice for each protein. By comparing nanomolar protein values determined by the Bradford method and by amino acid analysis, we obtained correction factors of 0.88 for tubulin and 1.14 for MTBR for Bradford analysis relative to a bovine serum albumin standard.


RESULTS

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 [^3H]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(1), m(2), and m(3) (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(1)m(3)m(3)] and MTBR[mm(2)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(2) (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(1) (VKSKIGSTDNIKYQPKGG) and m(3) (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(2) sequence and replaced it with an identical length copy of the third octadecapeptide repeat m(3). 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(1)m(3)m(3)] 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(1)m(3)m(3)] 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(2), and this permitted us to identify critical residues involved in MT binding. (^3)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(2) 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(2) 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[mm(2)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. (^4)To learn more about the self-association of MTBR and MTBR[mm(2)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[mm(2)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[mm(2) 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[mm(2)m] mutant containing additional copies of m(2)-like sequences in place of m(1) and m(3). 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[mm(2)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[mm(2)m] behaved as a much larger species shown with a best fit of M(r) 37,000 (dashed line), suggesting that upward of 90% of the mutant was dimeric.




DISCUSSION

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 [^3H]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(1)m(3)m(3)] 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(1)m(3)m(3)] 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(2) 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.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant GM-44823. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Health Science Center, Gainesville, FL 32610-0245. Tel.: 904-392-9410; Fax: 904-392-2953.

(^1)
The abbreviations used are: MT, microtubule; MTBR, microtubule-binding region; BSA, bovine serum albumin; MES, 2-(N-morpholino) ethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; MEM, MES-EGTA-magnesium buffer; MAP, microtubule-associated protein.

(^2)
R. L. Coffey and D. L. Purich, unpublished observations.

(^3)
A. M. Ainsztein and D. L. Purich, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Dr. Michael Bubb for carrying out the sedimentation equilibrium experiments, to Dr. Richard Kowalski for useful discussions of MAP-2 effects on microtubule dynamics, to Dr. Brian Cain for expert advice on site-directed mutagenesis, and to Farzin Foruhari for assisting with mutagenesis and sequencing.


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