Functionally specific binding regions of microtubule-associated protein 2c exhibit distinct conformations and dynamics

Abstract

Microtubule-associated protein 2c (MAP2c)1 is a 49-kDa intrinsically disordered protein regulating the dynamics of microtubules in developing neurons. MAP2c differs from its sequence homologue Tau in the pattern and kinetics of phosphorylation by cAMP-dependent protein kinase (PKA). Moreover, the mechanisms through which MAP2c interacts with its binding partners and the conformational changes and dynamics associated with these interactions remain unclear. Here, we used NMR relaxation and paramagnetic relaxation enhancement techniques to determine the dynamics and longrange interactions within MAP2c. The relaxation rates revealed large differences in flexibility of individual regions of MAP2c, with the lowest flexibility observed in the known and proposed binding sites. Quantitative conformational analyses of chemical shifts, small angle X-ray scattering (SAXS), and paramagnetic relaxation enhancement measurements disclosed that MAP2c regions interacting with important protein partners, including Fyn tyrosine kinase, plectin, and PKA, adopt specific conformations. High populations of poly-proline II and α-helices were found in Fyn-and plectin-binding sites of MAP2c, respectively. The region binding regulatory subunit of PKA consists of two helical motifs bridged by a more extended conformation. Of note, although MAP2c and Tau did not differ substantially in their conformations in regions of high sequence identity, we found that they differ significantly in long-range interactions, dynamics, and local conformation motifs in their N-terminal domains. These results highlight that the N-terminal regions of MAP2c provide important specificity to its regulatory roles and indicate a close relationship between MAP2c's biological functions and conformational behavior.Cytoskeletal microtubule associated proteins (MAPs) bind, stabilize, and regulate dynam- Conformation and Dynamics of MAP2cics of microtubules, in a phosphorylation-dependant manner. MAP2 and Tau are neuronal MAPs, MAP2 being expressed mainly in dendrites, whereas Tau is found in axons (1). Both MAP2 and Tau are expressed as different spliced variants. Tau isoforms expressed in human brain differ in the number of near-amino terminal inserts as well as in the number of repeats in the Microtubule Binding Domain (MTBD), whereas MAP2 isoforms differ in the length of the N-terminal projection domain (2, 3). Expression of both MAP2 and Tau isoforms is regulated during development. The high-molecular weight isoforms, MAP2a and MAP2b, contain 1830 amino-acids, and the two low molecular weight variants, MAP2c and MAP2d, consist of 467 and 498 amino acids, respectively. MAP2c is the smallest functional isoform, expressed mainly during embryonal brain development. After birth, its expression is restricted to regions exhibiting post-natal plasticity, such as the olfactory bulb (4), suggesting a role in neuronal development.Tau and MAP2c share a high sequence homology in the C-terminal part, containing MTBD, but differ in the N-terminal part, comprising the acidi...

Structure and Functions of Microtubule Associated Proteins Tau and MAP2c: Similarities and Differences

“…Formation of transient local structures discussed in this paper was inferred from NMR chemical shifts, measured in several studies [38,40,46,47,48,49,50,51,52,53,54,55], and converted to populations of conformers occupying different regions of the Ramachandran diagram by the ASTEROIDS algorithm [39,56,57]. The ASTEROIDS analysis also included results of NMR paramagnetic relaxation enhancements and small-angle X-ray scattering, describing long-range contacts and overall shapes of the studied molecules, respectively.…”

“…Occurrence of two secondary structure motifs that were present most often in the ensembles of conformers selected by the ASTEROIDS analysis (α-helix and polyproline II) is schematically depicted in Figure 2. To give more quantitative description of the propensity to form secondary structures, populations of continuous stretches of seven residues found in the same conformation in the selected ensembles are plotted for individual tau40 and MAP2c regions in Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7 [57]. Residues with increased populations of β-turn conformations [60] are also marked in Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7.…”

Fyn Regulates Binding Partners of Cyclic-AMP Dependent Protein Kinase A

“…In the case of MAP2, PKA-induced phosphorylation of MAP2 is known to disrupt the microtubule-MAP2 interaction and promote MAP2 localization to actin fibers in the cell periphery [34]. Interestingly, MAP2 is known to bind both PKA and Fyn [35,36], and PKA-induced phosphorylation of Fyn S21 targets Fyn to focal adhesions [19]. The finding that the MAP2/PKA interaction was decreased in the presence of Fyn, despite the detection of Fyn peptides in complex with PKA, suggests that PKA activity could promote dissociation of MAP2 from the PKA holoenzyme and microtubules.…”

Microtubule Dysfunction: A Common Feature of Neurodegenerative Diseases

“…The structural MAPs are probably the most intensely studied in the context of neurodegeneration. They are classified in two categories, the type I (MAP1a/1b family) and the type II (MAP2/tau family), with MAP2 and tau the major MAPs expressed in the central nervous system [ 168 ].…”

“…Tau is mainly found in axon of neurons where it promotes MT assembly and bundle formation [ 168 , 182 , 183 , 184 ]. In vitro experiments have demonstrated that tau promotes tubulin assembly and ring formation [ 185 ].…”