A common substrate recognition mode conserved between katanin p60 and VPS4 governs microtubule severing and membrane skeleton reorganization

Abstract

Katanin p60 (kp60), a microtubule-severing enzyme, plays a key role in cytoskeletal reorganization during various cellular events in an ATP-dependent manner. We show that a single domain isolated from the N terminus of mouse katanin p60 (kp60-NTD) binds to tubulin. The solution structure of kp60-NTD was determined by NMR. Although their sequence similarities were as low as 20%, the structure of kp60-NTD revealed a striking similarity to those of the microtubule interacting and trafficking (MIT) domains, which adopt anti-parallel three-stranded helix bundle. In particular, the arrangement of helices 2 and 3 is well conserved between kp60-NTD and the MIT domain from Vps4, which is a homologous protein that promotes disassembly of the endosomal sorting complexes required for transport III membrane skeleton complex. Mutation studies revealed that the positively charged surface formed by helices 2 and 3 binds tubulin. This binding mode resembles the interaction between the MIT domain of Vps4 and Vps2/CHMP1a, a component of endosomal sorting complexes required for transport III. Our results show that both the molecular architecture and the binding modes are conserved between two AAA-ATPases, kp60 and Vps4. A common mechanism is evolutionarily conserved between two distinct cellular events, one that drives microtubule severing and the other involving membrane skeletal reorganization.

FIGURE 1.

Domain architectures and multiple sequence alignment of kp60s and proteins containing MIT domains. A, domain architectures of mouse kp60, katanal1 and -2, and human Vps4b. The amino acid identities of each domain and full-length proteins between kp60 and other proteins are indicated. C.C., coiled-coil; MIT, MIT domain; LisH, LIS1 homology domain; AAA, AAA domain. B, multiple sequence alignment of kp60-NTDs and related proteins with secondary structure elements of kp60-NTD. The secondary structure elements are shown at the top of the figure. The α-helices (α1–3) are represented as thick lines and the C.C. region as a coil. Filled and open circles above the alignments indicate well conserved and less conserved core residues, respectively (see Fig. 2A). Triangles indicate residues substituted with Ala for examining tubulin binding. (Filled triangle, involved in tubulin binding; open triangle, not involved.) Protein names and UniProtKB accession numbers are as follows: kp60 human (O75449); kp60 mouse (Q9WV86); kp60 Drosophila (Q9VN89); kp60 Arabidopsis (Q9SEX2); katanal1 human (Q9BW62); katanal1 mouse (Q8K0T4); Vps4b human (O75351); Vps4b mouse (P46467); Vps4a human (Q9UN37); SNX15a human (Q9NRS6); spartin human (Q8N0X7); and spastin human (Q9UBP0). The sequence alignment was generated by ClustalX (62).

FIGURE 2.

Solution structure of kp60-NTD. A, stereo view of the best fit superposition of the 20 structures with lowest target functions. Side chains of buried residues with solvent accessibility less than 10% are shown (cyan). B, top, electrostatic surface potential mapped onto a van der Waals surface diagram. The color scale ranges between −20 k_BT (red) to +20 k_BT (blue), where k_B is Boltzmann's constant and T is temperature. Bottom, sequence conservation among the kp60-NTDs is mapped on the surface. Conservative and variable residues are colored purple and cyan, respectively. The color codes were produced by ConSurf (63). Ribbon diagrams of the kp60-NTDs are shown in the middle. The surface composed of helices 2 and 3 is shown as the front view (left) and the rear view (right).

FIGURE 3.

Structural comparisons of kp60-NTD with MIT domains. Ribbon diagrams of the proteins are as follows: A, kp60 (PDB code 2rpa); B, Vps4b (PDB code 1wr0); C, spastin (PDB code 3eab); D, spartin (PDB code 2dl1). Identity (top, %) and r.m.s.d. (bottom, Å) between kp60-NTD and the MIT domains are also presented. E, superposition of kp60-NTD (blue), Vps4b-MIT (magenta), spastin-MIT (pale green), and spartin-MIT (orange).

FIGURE 4.

Interactions of kp60-NTD with tubulin. A, pulldown assays of tubulin with GST-tagged kp60-NTDs of wild type and Ala mutants and Vps4b-MIT in vitro. Tubulin was used as the input. Molecular size is shown in lane 2. Only the buffer and the GST tag used as negative controls are shown in lanes 3 and 4. Recombinant proteins used for pulldown are indicated at the top of the gel. SDS-PAGE was silver-stained. B and C, side and top views of the ribbon diagram of kp60-NTD, respectively. Side chains of residues that were substituted with Ala are shown. In the pulldown assay, residues that were affected and unaffected by Ala mutations for tubulin binding are colored red and blue, respectively. D, top view of the ribbon diagram of the complex between Vps4-MIT and CHMP1a (yellow) (PDB code 2jq9). Side chains of the residues interacting between Vps4 and CHMP1a are indicated.

FIGURE 5.

Schematic diagram of architecture and molecular function similarities between kp60 and Vps4. kp60 catalyzes the disassembly of MT via N-terminal domain binding, which results in MT severing. Vps4 catalyzes the release of the ESCRT-III protomer via the MIT domain binding, which results in endosomal membrane invagination. For both biological events, the N-terminal domains serve as adaptors for the polymeric macromolecules, thereby disassembling either the cytoskeleton or the membrane skeleton in an ATP-dependent manner.

FIGURE 6.

Model of α-tubulin binding with kp60-NTD. Ribbon diagram of a model complex between kp60-NTD and a tubulin tetramer (gray) is shown. α-Tubulin helix 12, a putative interface of kp60-NTD, is colored yellow.