Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines

Abstract

Meiotic clade AAA ATPases (ATPases associated with diverse cellular activities), which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT (endosomal sorting complexes required for transport) pathway and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT (microtubule interacting and trafficking) domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and they appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops and that recruitment to the protein lattice via MIT domains also relieves autoinhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, as well as potentially katanin and fidgetin, are highly coordinated.

Keywords: ESCRT; Vps4; enzyme mechanism; microtubules; spastin.

Figure 1. Domain structures of meiotic AAA ATPases and their binding partners

Domains for which some structural information is available are color coded and labeled. MIT domain, cyan; large AAA ATPase domain (AAA-L), light blue; small AAA ATPase domain (-S), dark blue; β-domain, magenta; C-terminal helix (C), green; MTBD, dark red. Spastin isoform variants hydrophobic domain (HD) and exon 4 (4). The Vps4 binding partner LIP5/Vta1, wheat. The katanin p60 binding partner katanin p80 and WD40-less p80, white.

Figure 2. Substrates of meiotic clade AAA ATPases

(a) Structure of the heterodimer of GTP-bound α-tubulin (green/cyan) and GDP-bound β-tubulin (pink/magenta). The unstructured C-terminal tails that are engaged by the pore loops of spastin and katanin are colored red. (b) Schematic of the microtubule structure. Growing microtubules add GTP-bound tubulin heterodimers (green/blue) to their plus end. Following assembly, β-tubulin hydrolyses ATP to GDP (green/pink). C-terminal tails are shown for some representative subunits. (c) Crystal structure of CHMP3 (PDB ID: 3FRT) showing the canonical four helix bundle of the ESCRT-III fold (green). Sequences from helix 5 (red) are preferentially engaged by Vps4 pore loops. The MIM motif is part of the unstructured C-terminal tail and is shown schematically. (d) ESCRT-III assembles into membrane-bound helical polymers. MIM motifs are exposed on the surface, where they are available to recruit MIT-domain containing proteins, including Vps4.

Figure 3. MIM-MIT interactions

(a) Superposition of the MIT domains of human VPS4A (cyan, PDB ID: 2JQH), human spastin (pale yellow, PDB ID: 3EAB) and mouse katanin (rose, PDB ID: 2RPA). (b) Summary of known MIM-MIT interactions between human ESCRT-III proteins and the MIT domains of VPS4A, VPS4B, LIP5 and spastin^{; ; ; ; ; ; ; ; ; ;} . The type of interaction is indicated by shape. The affinity is indicated by fill color. Green outlines denote interactions that have been characterized structurally. “n.d.”, not determined. No ESCRT-III interactions have been reported for katanin or fidgetin. (c) Structure of the human VPS4A MIT domain in complex with the CHMP1B MIM1 (left, PDB ID: 2JQH) and the CHMP6 MIM2 (right, PDB ID: 2K3W). (d) Structure of the human spastin MIT domain in complex with the CHMP1B MIM3 (right, PDB ID: 3EAB). (e) Structure of MIM5-type binding of the C-terminus of CHMP5 to the tandem MIT domain of LIP5 (PDB ID: 2LXM).

Figure 4. Structure of the AAA ATPase cassette

Crystal structures of (a) human VPS4B (PDB ID: 1XWI), (b) human spastin (PDB ID: 3VFD) and (c) human fidgetin-like 1 (FIGL-1, PDB ID: 3D8B). Coloring is as in Figure 1. The N-terminal helix (red) in microtubule-severing enzymes lies within the sequence that has been mapped to contain the MTBD in spastin.

Figure 5. Hexameric models of spastin and Vps4

Models of p97-like hexamers were generated by superposition of the subunit structures of spastin (left) or VPS4B (right) on the D1 ring of the p97 hexamer (not shown). Hexamerization is favored by the avidity effect of MIT domains binding to the ESCRT-III and tubulin lattices. Spastin and other microtubule-severing enzymes bind microtubules through their MIT domains and microtubule binding domain (MTBD), as indicated by red arrows. The interaction between the LIP5 VSL domain and the VPS4B β-domain was modeled after the crystal structure of the equivalent yeast proteins.

Figure 6. Model of ATPase assembly and protein lattice disassembly

Meiotic clade AAA ATPases are monomeric or dimeric in the cytoplasm. The microtubule or ESCRT-III protein polymer (green) is depicted schematically. Recruitment of the ATPase by MIM-MIT interactions or microtubule interactions promotes formation of the active hexamer through increased local concentration. Substrate binding to the central pore loops requires an asymmetric conformation of the hexamer, which is induced by ATP hydrolysis. MIM-MIT interaction releases autoinhibition of substrate engagement at the Vps4 pore loops. Pore loops initially bind acidic motifs in the C-terminal tails of either ESCRT-III or tubulin, and translocation through the central pore results in subunit unfolding and consequent disassembly of the ESCRT-III lattice or severing of microtubules.