Structural Basis for the Association of MAP6 Protein with Microtubules and Its Regulation by Calmodulin

Background: Microtubules are cold-sensitive, but some cold-stable microtubules are observed in specific cells due to the presence of MAP6. Results: Structural data detail how a MAP6 fragment stabilizes microtubules and how calmodulin regulates its activity. Conclusion: MAP6 may stabilize microtubules by bridging adjacent tubulin heterodimers, an activity sterically hindered by calmodulin. Significance: This work provides a better understanding of cellular microtubule stabilization and its regulation by calmodulin. Microtubules are highly dynamic αβ-tubulin polymers. In vitro and in living cells, microtubules are most often cold- and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold- and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90–177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90–177) to microtubules with a 1:1 MAP6(90–177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90–177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca2+-calmodulin competes with microtubules for MAP6(90–177) binding and that the binding mode of MAP6(90–177) to microtubules and Ca2+-calmodulin involves a common stretch of amino acid residues on the MAP6(90–177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca2+-calmodulin.

Microtubules are highly dynamic ␣␤-tubulin polymers. In vitro and in living cells, microtubules are most often cold-and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold-and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90 -177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90 -177) to microtubules with a 1:1 MAP6(90 -177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90 -177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca 2؉ -calmodulin competes with microtubules for MAP6(90 -177) binding and that the binding mode of MAP6(90 -177) to microtubules and Ca 2؉ -calmodulin involves a common stretch of amino acid residues on the MAP6(90 -177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca 2؉ -calmodulin.
In certain cell types such as neurons, fibroblasts, or glial cells, a subpopulation of microtubules (MTs) 4 remains stable in depolymerizing conditions such as exposure to the cold or depolymerizing drugs (1)(2)(3)(4)(5). Search for cellular factors responsible for this stabilization led to the discovery of a class of microtubule-associated proteins named MAP6 (also called STOP) (6 -8). In mammals, MAP6 isoforms are encoded by a single gene (map6) and result from mRNA splicing and alternative promoter use (9 -11). The physiological role of MAP6 proteins is not yet fully understood, but phenotypic and cellular analyses of MAP6 null mice indicated that MAP6 proteins are involved in a number of neuronal functions. MAP6 null mice present defects in synaptic plasticity and neurotransmission associated with severe behavioral disorders (12). Interestingly, these disorders can be alleviated by drugs similar to neuroleptics, suggesting that these mice may represent a valuable animal model for the study of schizophrenia (13). In line with these findings, genetic studies have pointed out a possible link between MAP6 and schizophrenia (14), suggesting that map6 could be considered as a candidate gene that predispose to schizophrenia and thus be used as a potential biomarker for its early detection (15,16). Besides, MAP6 proteins were also found to be implicated in adult olfactory neurogenesis (17), and MAP6-F (the fibroblastic MAP6 isoform) was proposed to be a temperature sensor that protects MTs from temperature variations in animals during episodes of torpor or hibernation (18).
MAP6-N (the neuronal MAP6 isoform) is the largest MAP6 isoform and is mainly expressed in mature neurons. This 952residue protein contains several repeated motifs called Mn and Mc modules, which are linked to its ability to stabilize MTs (see Fig. 1A) (19). The Mn modules allow MAP6 to protect MTs against cold-and nocodazole-induced depolymerization while the Mc modules permit MAP6 to stabilize MTs in cold condition. MAP6-N contains two consecutive Mn modules in its N-terminal part (Mn1 and Mn2) and an additional one isolated in the middle of its sequence (Mn3). The Mc modules are clustered in the central domain as five highly conserved repetitions organized in tandem. Whereas Mn modules are well conserved across species (see Fig. 1B), the homology between Mn1, Mn2 and Mn3 is low, and they share only a few residues (Fig. 1C). It is worth noting that the number and the organization of Mn and Mc modules can be different among the MAP6 isoforms, and this may lead to functional variations toward the propensity of MAP6 proteins to stabilize MTs (8,18,19). In addition, it was shown that the MT stabilization activity of MAP6 proteins is inhibited by calmodulin (CaM) (20,21). Accordingly, peptide arrays revealed that MAP6-N possesses several CaM binding sites, some of which being found in the Mn and Mc modules, suggesting that these modules have a dual propensity to interact both with MTs and CaM (19). Very few studies addressed the structural basis of the interaction of MAP6 with MTs and of its interplay with CaM. An NMR study of the interaction between CaM and a peptide model corresponding to the consensus Mc motif showed an unusual interaction mode with a very limited structural impact on the CaM structure (22).
In the present work, we undertook biochemical and structural analyses of the interaction of MAP6-N with MTs and CaM to enlighten the mechanism by which it stabilizes MTs and the mechanisms by which such stabilization is regulated by CaM.
As MAP6-N has a high molecular weight, we focused this work on a minimal MAP6-N fragment (residues 90 -177, named herein MAP6(90 -177)), which recapitulates the MT stabilization and CaM binding properties of MAP6 proteins. We show here that the MAP6(90 -177) fragment, which comprises the Mn1 and Mn2 modules, interacts tightly with MTs, and has a powerful MT stabilization activity in vitro and in cultured cells. It also interacts with CaM in a Ca 2ϩ -dependent manner. Using a combination of biochemical and NMR experiments, we determined at the residue scale the location of its MT and CaM binding sites, and we clearly demonstrated that Ca 2ϩ -CaM binding impairs MAP6(90 -177) association with MTs probably because the Ca 2ϩ -CaM binding sites substantially overlap the MT binding sites. Furthermore, we examined by NMR the structural impact of MAP6(90 -177) binding on the Ca 2ϩ -CaM structure. The results showed dramatic conformational and dynamical changes of Ca 2ϩ -CaM affecting its overall structure which led us to propose a binding mode for Ca 2ϩ -CaM on the Mn modules of MAP6 proteins.
Tubulin was purified from sheep brain using the method of Castoldi and Popov (23) and stored in liquid nitrogen. Before use, an additional cycle of polymerization/depolymerization was performed, and tubulin was resuspended in 50 mM MES-KOH, 4.1 M glycerol, 1 mM MgCl 2 , 0.5 mM EGTA, pH 6.9. Tubulin concentration was determined by spectrophotometry using an extinction coefficient ⑀ 278 nm ϭ 1. To verify the integrity of the tubulin after the cold treatment, the temperature was then set again at 37°C. The presence of MTs after the cold shock was examined by atomic force microscopy (AFM) on 10-l aliquots containing 35 M MAP6(90 -177) and sampled at t ϭ 30 min. The samples were deposited on freshly cleaved mica and dried for AFM imaging as described previously (24). All AFM experiments were performed in intermittent mode with a multimode AFM instrument (Digital Instruments, Veeco, Santa Barbara, CA) operating with a Nanoscope IIIa controller (Digital Instruments). Images were collected at a scan frequency of 1.5 Hz and a resolution of 512 ϫ 512 pixels.
Immobilized CaM-binding Assay-CaM-agarose beads (Sigma-Aldrich) were equilibrated in 50 mM MES-KOH, 1 mM CaCl 2 , pH 6.9, and incubated with pure MAP6(90 -177) for 30 min at 20°C. Beads were then washed with the same buffer. Flow-through, washes, and CaM bead fractions were analyzed on SDS-PAGE, and the proteins were revealed by Coomassie Blue staining. The Ca 2ϩ -dependence of this interaction was tested with calcium-free buffer supplemented with 5 mM EGTA.
Isothermal Titration Calorimetry (ITC)-ITC measurements were performed at 25°C using an ITC 200 calorimeter (GE Healthcare). Samples were thoroughly degassed before each titration. Titration and sample solutions were prepared in 20 mM MES-KOH, 50 mM NaCl, 1 mM DTT either with 1 mM CaCl 2 or 5 mM EGTA, pH 6.9. Titrations were carried out by injecting 25 consecutive aliquots (1.5 l) of 200 M MAP6(90 -177) into the ITC cell (0.2 ml) containing 25 M CaM. A background titration consisting of the identical titration solution but containing only the buffer in the cell was subtracted from each titration to quantify heat produced by sample dilution. The value obtained was subtracted from the heat of reaction to give the effective heat of binding. The resulting titration data were analyzed using the ORIGIN software (MicroCal, Inc.). The molar binding stoichiometry, binding constant (K a ϭ 1/K d ) and binding enthalpy (⌬H) were determined by fitting the binding isotherm to a model with one set of sites. For the fit, no constraint in stoichiometry, K a and ⌬H was introduced. Changes in free energy (⌬G) and in entropy (T⌬S) were calculated from ⌬G ϭ ϪRT ln K a ϭ ⌬H Ϫ T⌬S, where R is the gas constant and T is the temperature in Kelvin.
A Ca 2ϩ -loaded uniformly 15 N-13 C-labeled CaM sample was prepared at 60 M in 18 mM Tris-HCl, pH 7.8, and 10% D 2 O and studied in a Shigemi tube (Shigemi, Inc., Allison Park, PA). The formation of the MAP6(90 -177)⅐Ca 2ϩ -CaM complex was examined on samples by direct addition of different aliquots of a concentrated solution of unlabeled MAP6(90 -177) (from a 0.6 mM stock solution) to the 15 N-13 C-labeled Ca 2ϩ -CaM NMR sample to reach 1.5:1 MAP6(90 -177):Ca 2ϩ -CaM stoichiometry. NMR spectra were acquired at 27°C on an Agilent VNMRS 800 MHz spectrometer using a cryoprobe. A BEST-TROSY experiment was used as it provided resolution without losing sensitivity compared with 1 H-15 N HSQC (27). 128 ϫ 930 complex points in the F1 and F2 dimensions were acquired for an experimental time of ϳ3 h. All data were processed using the NMRPipe software (28). Figures were represented with VMD software (29).  (Fig. 2). As MAP6(90 -177) thus binds to and stabilizes MTs in a cellular context, the investigation of its biochemical and structural properties appears of interest for the characterization of the full length MAP6 proteins.

MAP6(90 -177) Induces MT Resistance to Cold and Nocoda
MAP6(90 -177) Binds to MTs and Induces MT Resistance to Cold in Vitro-As a second step, to distinguish between a direct and indirect binding through putative protein partners, we analyzed the association of MAP6(90 -177) with MTs in vitro using co-sedimentation experiments with taxol-stabilized MTs. In the absence of MT, MAP6(90 -177) was not detectable in the pellet, demonstrating that MAP6(90 -177) is soluble in these conditions. When MAP6(90 -177) was incubated with increasing amounts of MTs, a concentration-dependent MAP6(90 -177) binding to MTs was observed (Fig. 3A). This result shows that MAP6(90 -177) binds directly to MTs. In addition, we found that shearing MTs, which increases the number of MT ends, did not significantly affect MAP6(90 -177) binding to MTs (data not shown). To further characterize the MAP6(90 -177)/MT interaction, MAP6(90 -177) at different concentrations was then incubated with a fixed amount of taxol-stabilized MTs (Fig. 3B). MTs were then pelleted, and the MAP6(90 -177) content was analyzed by SDS-PAGE and quantified. The results show that the binding of MAP6(90 -177) to MTs is saturable with an estimated K d of 6 M and a 1:1 MAP6(90 -177):tubulin heterodimer stoichiometry.
We then assessed the ability of the MAP6(90 -177) fragment to stabilize MTs against cold. After polymerization of tubulin into MTs at 37°C, MTs were exposed to cold (ice for 10 min) in the absence or presence of MAP6(90 -177). SDS-PAGE analysis shows that the amount of MTs in the pellet increases dra-matically in the presence of MAP6(90 -177) after cold treatment (Fig. 3C). Similar results were observed whether the MAP6(90 -177) fragment was mixed to tubulin before polymerization or added to assembled MTs. To confirm that the MAP6(90 -177) fragment truly protected MTs from cold depolymerization, we examined the dynamics of MT assembly by turbidimetry at 370 nm (Fig. 3D). Results show that the kinetics of tubulin polymerization are very similar in the absence or presence of different concentrations of MAP6(90 -177), demonstrating that MAP6(90 -177) has no impact on the nucleation and elongation steps. In addition, the steady state turbidity value was also similar in both cases suggesting that no particular supra-assembly of MTs such as bundles were formed in the presence of MAP6(90 -177). The cold shock was performed at steady state. In the control condition, it induced a clear MT depolymerization. The effect of this cold shock diminished dramatically in the presence of MAP6(90 -177), and a total protection was observed when the MAP6(90 -177): tubulin heterodimer ratio reached a value of about 1:1. To ascertain that the macromolecular objects diffusing light during the cold shock in the presence of MAP6(90 -177) are really MTs, we performed AFM imaging which shows filamentous structures with length and width in good agreement with MT dimensions (Fig. 3E) (31).
Quantification of the MAP6(90 -177)/CaM Interaction-It was previously demonstrated that MAP6-N binds to CaM (8) and that Mn modules are implicated in part in this interaction (19). To further investigate such interaction, we examined the binding of MAP6(90 -177) to CaM immobilized on beads (Fig.  4A). The results show that MAP6(90 -177) is able to bind to CaM but only in the presence of CaCl 2 . Indeed, when the binding experiment was realized with 5 mM EGTA instead of CaCl 2 , no binding was observed, demonstrating that calcium is necessary for this interaction.
We next carried out ITC experiments to quantify the MAP6(90 -177)/CaM interaction. Fig. 4B displays the binding isotherm of MAP6(90 -177) titration into the Ca 2ϩ -CaM solution. The best fit of the integrated isotherm indicated that one MAP6(90 -177) binds to two Ca 2ϩ -CaM molecules with a K d value of 0.4 M (Fig. 4C). The same ITC experiment carried out with EGTA in place of CaCl 2 showed no detectable interaction.
Identification of MAP6(90 -177) Residues Implicated in the Interaction with MTs and Ca 2ϩ -CaM-Large amounts of pure and soluble MAP6(90 -177) could be obtained from recombinant expression in Escherichia coli, which allowed NMR study of MAP6(90 -177) interactions with its partners at the residue scale. The 1 H-15 N HSQC spectrum of MAP6(90 -177) in aqueous buffer shows well resolved resonances with a weak dispersion of correlation peaks (Ͻ1 ppm and ϳ20 ppm on the 1 H and 15 N dimensions, respectively), suggesting that under these conditions, the MAP6(90 -177) does not fold into any defined tertiary structure (Fig. 5A). NMR assignment of the backbone resonances of MAP6(90 -177) was achieved (supplemental Table  1). Analysis of the chemical shifts deviations from random coil values of 13 CO and 13 C␣ (32) also indicated that MAP6(90 -177) does not contain significant secondary structure (Fig. 5B). In agreement with this, we observed that during the course of overexpression and purification, the MAP6(90 -177) fragment exhibited a high sensitivity to protease degradation together with a high thermostability, properties that are frequently observed for intrinsically unstructured proteins.
To identify MAP6(90 -177) residues that mediate MT and Ca 2ϩ -CaM interaction, 1 H-15 N HSQC spectra of free 15 N-MAP6(90 -177) and bound to unlabeled tubulin, MTs and Ca 2ϩ -CaM were recorded (Fig. 6A). Because of the large size of these complexes, many 1 H-15 N cross-peaks of residues implicated in the interaction underwent line broadening and disappeared, which helped us to point them out. Experiments carried out either with tubulin or MTs gave identical results, whereas control experiments performed with BSA showed no modification. A peak by peak analysis using assignment of MAP6(90 -177) allowed us to define two MT binding sites delimited by residues 121-138 and 150 -175 (Fig. 6A). As the C-terminal tail of tubulin is a major binding site for many MT partners, we tested whether this part of tubulin is implicated in the MAP6(90 -177)/MT interaction. Co-sedimentation assays were carried out in the presence of an excess of a recombinant tubulin fragment corresponding to the C-terminal tail of ␣-tubulin (␣Tub410C) (supplemental Fig. S1) (33). Results show that this ␣Tub410C fragment did not compete with MTs for MAP6(90 -177) binding. This result was confirmed by NMR, which showed that the 1 H-15 N HSQC spectrum of 15 N-MAP6(90 -177) remained identical in the absence or in the presence of an excess of unlabeled ␣Tub410C (data not shown). We then attempted to locate the Ca 2ϩ -CaM binding site on MAP6(90 -177) using similar 1 H-15 N HSQC mapping experiments (Fig. 6A). As seen for MTs, Ca 2ϩ -CaM binding to 15   for MT cold depolymerization was tested in the presence of Ca 2ϩ -CaM (Fig. 6C). In this experiment, Ca 2ϩ was required to obtain the holo-form of CaM. However, due to the destabilizing effect of Ca 2ϩ on MTs, experiments were performed in the presence of excess of spermine, which acts as a Ca 2ϩ -competitive inhibitor for MT binding (33). Polymerized MTs were incubated on ice alone (control, the two left lanes) or with MAP6(90 -177) (the two middle lanes) or with MAP6(90 -177) and Ca 2ϩ -CaM (the two right lanes). Results show clearly that MAP6(90 -177) no longer protects MTs in the presence of Ca 2ϩ -CaM.
Binding of MAP6(90 -177) to Ca 2ϩ -CaM Induces Large Conformational Changes of the Ca 2ϩ -CaM Structure-To study the structural impact of MAP6(90 -177) binding on the Ca 2ϩ -CaM structure, we carried out NMR experiments with purified and 15 N-13 C-labeled Ca 2ϩ -CaM. BEST-TROSY spectra of 15 N-13 C-Ca 2ϩ -CaM in the absence and presence of unlabeled MAP6(90 -177) were recorded (Fig. 7A). The addition of MAP6(90 -177) to Ca 2ϩ -CaM resulted in very large changes of the spectrum profile of Ca 2ϩ -CaM with modifications of the peaks number and of the chemical shifts distribution. Residues affected by this interaction are reported on the CaM sequence and on a crystallographic structure of Ca 2ϩ -CaM (Fig. 7, B and C) (34). Results show that the perturbations are found mostly in the N-terminal domain and in the central ␣-helix of Ca 2ϩ -CaM. Indeed, NMR mapping points out a first stretch in the N-terminal domain (residues 13-39) and a second long stretch comprising the end of the N-terminal domain and more than half of the central ␣ helix (residues 49 -80). The C-terminal domain is affected but in a lesser extent. Together, these observations indicate that the binding of MAP6(90 -177) induces wide range conformational and dynamical changes in the Ca 2ϩ -CaM structure, implicating mostly its N-terminal domain and the central ␣-helix.
MTs and Ca 2ϩ -CaM Compete for the Same Binding Site on the Mn1 Module-We finally examined the possibility that a single Mn module could interact with MTs and Ca 2ϩ -CaM. To that end, we overexpressed and purified a 53-amino acid residue fragment encompassing the N-terminal part and the Mn1 module of MAP6(90 -177) (MAP6-N residues 90 -142, named herein MAP6(90 -142)). We observed that the 1 H-15 N HSQC spectrum of MAP6(90 -142) superimposes very well with that of the MAP6(90 -177) fragment, which strongly facilitated its assignment. When the MAP6(90 -142) fragment was studied in the presence of tubulin or MTs, we observed on its 1 H-15 N HSQC spectrum (Fig. 8A) line broadening responsible for the disappearance of some peaks corresponding to the stretch of residues 121-138 with the exception of Cys-135. (Fig. 8B). Interestingly, the 1 H-15 N HSQC spectrum of MAP6(90 -142) acquired in the presence of Ca 2ϩ -CaM (Fig. 8A) gave very close results with line broadening and disappearance of peaks corresponding to residue Val-118 and of the stretch 121-138 except for Gln-129 and Ser-134 (Fig. 8B). Hence, these results show that the Mn1 module, when considered isolated from the other Mn modules, is able to bind to both MTs and Ca 2ϩ -CaM in a similar manner.

DISCUSSION
MAP6(90 -177) Binds and Stabilizes MTs-The aim of this work was to better understand the molecular mechanisms underlying the stabilization of MTs by MAP6 and its regulation by CaM using biochemical and structural investigations. To that end, we took advantage of a MAP6-N fragment encompassing its Mn1 and Mn2 modules, which recapitulates the functional properties of the full-length protein. As a first step, we studied the interaction between MAP6(90 -177) and MTs. Using co-sedimentation assays, we found that MAP6(90 -177) interacts with MTs with an apparent K d value of ϳ6 M and a 1:1 MAP6(90 -177):tubulin heterodimer stoichiometry (Fig.  3B). To the best of our knowledge, this is the first report on a K d value regarding the interaction of Mn modules with MTs. Del- This suggests that the binding of MAP6 to MTs may be cooperative. Co-sedimentation assays (supplemental Fig. S1) and NMR experiments (data not shown) using a ␣-tubulin C-terminal fragment (␣Tub410C, residues 410 -451 from ␣ 1a -tubulin (33)) indicated that this part of tubulin is not sufficient for the interaction with MAP6(90 -177). This agrees with previous report showing that, unlike other MAPs, MAP6 can bind significantly to any tubulin isoform whatever its degree of C-terminal modification by polyglutamylation (35).
We observed that the stabilization of MTs against cold and nocodazole by MAP6(90 -177) does not induce MT aggregates or bundles in contrast to what was observed with full-length MAP6-N by immunofluorescence microscopy (7). In the latter case, the formation of MT bundles may be due either to the presence of multiple Mn and Mc repeats or to a possible multimerization of the protein via stretches of amino acid residues that are located outside of the MAP6(90 -177) region. We next mapped the MAP6(90 -177) interaction with MTs to two MAP6(90 -177) binding sites defined by residues 121-138 and 150 -175, which correlate strongly with Mn1 and Mn2 modules, respectively (19). Interestingly, NMR data show that a single Mn module (MAP6(90 -142)) can still interact with MTs, suggesting that each Mn module in MAP6 may serve as anchor point for MT binding. Accordingly, each Mn repeat may recognize a conserved binding motif located on ␣and ␤-tubulin. From a structural point of view, MT stabilization by MAP6(90 -177) is thus likely to occur through the reinforcement of the longitudinal contacts between tubulin heterodimers within a protofilament as seen for Tau (36) or lateral contacts between adjacent tubulin heterodimers, preventing protofilament separation similar to the stabilization mode of doublecortin (37). In addition, we cannot exclude that MAP6(90 -177) may also stabilize MTs by preventing cold-or nocodazole-induced conformational changes in tubulin by a more complex mechanism.
Association of MAP6(90 -177) with MTs Is Regulated by Ca 2ϩ -CaM-Biochemical and ITC experiments showed that MAP6(90 -177) binds to CaM in a Ca 2ϩ -dependent manner with a K d value of 0.4 M and a 1:2 MAP6(90 -177):Ca 2ϩ -CaM stoichiometry (Fig. 4). This K d value lies in the range of that previously reported for MAP6-F/Ca 2ϩ -CaM (K d ϳ 1.7 and 8.1 M) (19) and In agreement with this hypothesis, we demonstrated for the first time using co-sedimentation assays that Ca 2ϩ -CaM is responsible for a stoichiometric inhibition of MAP6(90 -177) binding to MTs, resulting in a loss of MT stabilization. Altogether, these observations suggest that the regulation of MAP6 binding to MTs by Ca 2ϩ -CaM is a dynamic process rapidly modulated by the Ca 2ϩ charge state of CaM. A similar mechanism was proposed for Tau in the flip-flop model (39). Finally, this part of MAP6-N also comprises Ser-139, which can be phosphorylated by the Ca 2ϩ -calmodulin-dependent protein kinase II, which triggers the translocation of MAP6 from MTs to actin (40). Interestingly, this kind of regulation has already been observed for the microtubule-associated protein p35, which is also regulated by site-specific phosphorylations and Ca 2ϩ -CaM with MT overlapping binding sites (41). Similar mechanisms of CaM regulation were again suggested for Tau (42)(43)(44).
Impact of MAP6(90 -177) on Ca 2ϩ -CaM Structure, Implication for the Binding Mode-Previous NMR, infrared spectroscopy and differential scanning calorimetry investigations on the interaction of Ca 2ϩ -CaM with a model peptide corresponding to a single Mc motif revealed an unusual binding mode. Indeed, these studies showed that the Mc peptide interacts with the C-terminal domain of CaM, leaving it in an extended conformation with no significant structural change (22,38). The present data provide strong evidences that the binding mode of MAP6(90 -177) to Ca 2ϩ -CaM is distinct from that previously observed with Mc modules. However, the present NMR data show that the binding of MAP6(90 -177) to Ca 2ϩ -CaM produces large structural and dynamical changes in Ca 2ϩ -CaM. Interestingly, sequence analysis of the two sites of MAP6(90 -177) that interact with Ca 2ϩ -CaM suggests that they belong to the 1-5-10 class of canonical CaM-binding motifs found in several CaM-regulated proteins such as Ca 2ϩ -calmodulin-dependent protein kinase II and synapsin I (Fig. 9) (45,46). This class refers to a group of Ca 2ϩ -CaM partners whose key hydrophobic bulky residues are spaced by eight residues with an additional anchoring residue in the middle. Upon binding, both Ca 2ϩ -CaM and the target peptide motif undergo large conformational rearrangements. The canonical CaM-binding motif adopts an ␣-helical conformation and the two domains of CaM wraps around it, enclosing it in a hydrophobic channel within the globular core. The data obtained here on MAP6(90 -177)/ Ca 2ϩ -CaM interaction argue for a similar binding mode: (i) Ca 2ϩ is required for MAP6(90 -177) binding, a fact that is systematically observed with this type of binding mode; (ii) large structural modifications of the Ca 2ϩ -CaM structure are observed affecting its two domains and its central flexible helix; and (iii) the secondary structure prediction of MAP6(90 -177) show that residues 117-125 and 148 -158 may fold into ␣-helices. These stretches overlap the 1-5-10 motifs of MAP6(90 -177), and it is possible that they locally fold when in complex  (52)) are labeled. B, sequence of CaM showing the residues affected by the interaction (boldface type). The proline Pro-67 residue was arbitrarily included. The central ␣-helix separating the N-and C-terminal domains of CaM is underlined. C, spatial representations of the Ca 2ϩ -CaM structure (Protein Data Bank code 1OSA). The image on the right was obtained after a 180°rotation around the x axis and a 90°rotation around the z axis. Residues that are not significantly affected by the interaction are rendered as gray rigid blocks. Yellow spheres, Ca 2ϩ ions.
with Ca 2ϩ -CaM. It is worthy to note that neither the Mc modules nor the Mn3 module of MAP6 (which bind to Ca 2ϩ -CaM) contain such a 1-5-10 motif. This may explain previous reports showing an unusual binding mode for these CaM-binding sites (22). 5 Interestingly, some 1-5-10 motifs were shown to be implicated both in Ca 2ϩ -CaM and actin binding (47)(48)(49), and MAP6-N was shown to interact with actin (40). It would be of interest to probe whether this region is implicated in MAP6-N association with actin and whether Ca 2ϩ -CaM binding to this region may regulate such process.
In conclusion, we report here for the first time structure and functional information on a short region from MAP6 that recapitulates its ability to bind and stabilize MTs upon exposure to cold or nocodazole. The binding of this region to MTs appears modulated by a competitive interaction with Ca 2ϩ -CaM, which further documents the complex regulation of MT assembly by MAP6.