Structural insight into microtubule stabilization and kinesin inhibition by Tau family MAPs

Abstract

The Tau family microtubule-associated proteins (MAPs) promote microtubule stabilization and regulate microtubule-based motility. They share the C-terminal microtubule-binding domain, which includes three to five tubulin-binding repeats. Different numbers of repeats formed by alternative splicing have distinct effects on the activities of these proteins, and the distribution of these variants regulates fundamental physiological phenomena in cells. In this study, using cryo-EM, we visualized the MAP4 microtubule complex with the molecular motor kinesin-1. MAP4 bound to the C-terminal domains of tubulins along the protofilaments stabilizes the longitudinal contacts of the microtubule. The strongest bond of MAP4 was found around the intertubulin-dimer interface such that MAP4 coexists on the microtubule with kinesin-1 bound to the intratubulin-dimer interface as well. MAP4, consisting of five repeats, further folds and accumulates above the intertubulin-dimer interface, interfering with kinesin-1 movement. Therefore, these cryo-EM studies reveal new insight into the structural basis of microtubule stabilization and inhibition of kinesin motility by the Tau family MAPs.

Graphical abstract

Figure 1.

MAP4 and kinesin-1 can be simultaneously bound to the microtubules. (A) Primary structure of MAP4 and its microtubule-binding fragments 5R-MAP4 and 4R-MAP4. (B–G) Microtubule cosedimentation assays. All experiments were performed three independent times, and the concentrations of bound protein were calculated from each SDS-PAGE gel. Error bars in graphs represent SD (n = 3). (B) Typical SDS-PAGE gels of microtubule-binding assays of 4R- or 5R-MAP4 without kinesin-1. (C) Concentrations of bound MAP4 fragments when 16 µM 4R- or 5R-MAP4 was incubated with microtubules (in the absence of kinesin-1). There was no significant difference between the bound 4R- and 5R-MAP4. (D) Typical results for binding of 4R- or 5R-MAP4 to microtubules in the presence of 20 µM kinesin-1. (E) The concentrations of bound 4R- or 5R-MAP4 and kinesin-1 in the presence of 20 µM kinesin-1. The concentrations of bound 4R- or 5R-MAP4 in the absence of kinesin-1 are also shown. (F) Typical results of microtubule-binding assays of kinesin-1 with 16 µM 4R- or 5R-MAP4. Sup, supernatant; ppt, precipitate. (G) The concentrations of bound kinesin-1 and 4R- or 5R-MAP4. The concentrations of bound kinesin-1 in the absence of MAP4 are also shown.

Figure 2.

Cryo-EM reconstructions of MAP4–kinesin-1–microtubule complex. (A and D) Cryo-EM reconstruction of 5R-MAP4–kinesin-1–microtubule complex. Green, α-tubulin; light blue, β-tubulin; pink, kinesin-1; purple, residual densities (5R-MAP4). (B and E) Cryo-EM reconstruction of 4R-MAP4–kinesin-1–microtubule complex. Yellow, residual densities (4R-MAP4). (C and F) Cryo-EM reconstruction of kinesin-1–microtubule complex. Red, residual densities. (A–C) Cryo-EM reconstructions observed from the outer surface of microtubules. (D–F) Cryo-EM reconstructions observed from the inside of microtubules.

Figure 3.

Structural detail of MAP4–kinesin-1–microtubule binding. (A) Cryo-EM reconstruction of 5R-MAP4–kinesin-1–microtubule complex seen from the right side of the protofilament (left) and from the surface (right). Green, α-tubulin; light blue, β-tubulin; pink, kinesin-1; purple, 5R-MAP4. Higher threshold density for MAP4 is shown in opaque. (B) Cryo-EM reconstruction of 4R-MAP4–kinesin-1–microtubule complex. Green, α-tubulin; light blue, β-tubulin; pink, kinesin-1; yellow, 4R-MAP4. Higher threshold density for MAP4 is shown in opaque. (C) Magnified view of black dotted rectangle in A shown with Tau (blue; PDB ID: 6CVN). A 180°-rotated view is also presented. (D) Magnified view of red dotted rectangle in A shown with Tau. The anchor point sequences of Tau and corresponding sequence of MAP4 are also presented. (E) Amino acid sequences of the tubulin-binding repeat of bovine 5R-MAP4 and 4R-MAP4.

Figure 4.

Structural model of microtubule binding of MAP4 and kinesin inhibition by MAP4. MAP4 is bound to the microtubule through the anchor point around the interdimer interface and through the weak binding site around the intradimer interface. Kinesin-1 competes against MAP4 for the weak binding site and finally eliminates MAP4 from the weak binding site. MAP4 is still anchored at the anchor point, and the rest of the regions may fold and accumulate above the interdimer interface.