The AAA ATPase Vps4 binds ESCRT-III substrates through a repeating array of dipeptide-binding pockets

Abstract

The hexameric AAA ATPase Vps4 drives membrane fission by remodeling and disassembling ESCRT-III filaments. Building upon our earlier 4.3 Å resolution cryo-EM structure (Monroe et al., 2017), we now report a 3.2 Å structure of Vps4 bound to an ESCRT-III peptide substrate. The new structure reveals that the peptide approximates a β-strand conformation whose helical symmetry matches that of the five Vps4 subunits it contacts directly. Adjacent Vps4 subunits make equivalent interactions with successive substrate dipeptides through two distinct classes of side chain binding pockets formed primarily by Vps4 pore loop 1. These pockets accommodate a wide range of residues, while main chain hydrogen bonds may help dictate substrate-binding orientation. The structure supports a ‘conveyor belt’ model of translocation in which ATP binding allows a Vps4 subunit to join the growing end of the helix and engage the substrate, while hydrolysis and release promotes helix disassembly and substrate release at the lagging end.

Structures, Functions, and Dynamics of ESCRT-III/Vps4 Membrane Remodeling and Fission Complexes

“…Vps4 enzymes contain three different structural elements: ( a ) an N-terminal MIT domain that binds the tails of ESCRT-III proteins (Figures 2 c and 5 a ); ( b ) a central ATPase cassette comprising large and small domains that mediate hexamerization and ATP hydrolysis; and ( c ) a β-domain insert within the small ATPase domain that binds LIP5 (Vta1), an ATPase activator and ESCRT-III-binding protein. Our understanding of the enzyme’s structure and molecular mechanism has increased dramatically with recent high-resolution cryo-EM structures of Vps4 proteins in their active, hexameric ring conformations (Han et al 2017, Monroe et al 2017, Su et al 2017, Sun et al 2017), particularly structures of the yeast Vps4 enzyme in complex with an ESCRT-III substrate (Han et al 2017, Monroe et al 2017). These structures revealed that Vps4 forms an asymmetric ring hexamer that binds the exposed tails of ESCRT-III subunits and translocates the subunits through the constricted central pore of the hexamer.…”

“…The E subunit is displaced slightly from its idealized helix position, and the sixth (F) Vps4 subunit sits outside the helix and is fully disengaged from the substrate (Figure 5). The nucleotide state of each subunit in the active hexamer has not been determined with absolute certainty, but it appears that subunits A–C bind ADP•BeF x in an ATP-like configuration and D and E bind ADP, while the F subunit appears to lack a nucleotide (Han et al 2017). Nucleotides bind at subunit interfaces and are canonically coordinated by ( a ) a Walker A motif, which contacts the adenosine base, sugar, and first two phosphates; ( b ) a Walker B motif, which chelates a magnesium ion and catalytic water; and ( c ) two arginine finger residues from an adjacent subunit, which coordinate the gamma phosphate and help promote ATP hydrolysis (Wendler et al 2012).…”

“…ESCRT-III substrate engagement activates the enzyme by relieving autoinhibitory MIT domain interactions (Babst et al 2011, Han et al 2015, Merrill & Hanson 2010). The Vps4 hexamer can then assemble about ESCRT-III elements located upstream of the terminal MIT binding site (Han et al 2015, 2017; Monroe et al 2017; Shim et al 2008), yielding an active holoenzyme.…”

“…The ESCRT-III substrate binds in an extended (β-strand) conformation (Han et al 2017). Alternating side chains project from opposite sides of the strand, creating a helical array of repeating dipeptide units.…”

“…Structures are reproduced from Han et al (2017) (ATPase cassette) and Obita et al (2007) (MIT domain), ( b ) Top view of the asymmetric yeast Vps4 ring hexamer (subunits A–F) with associated dimeric VSL domains from Vta1 ( beige ), ESCRT-III peptide ( green ), and nucleotides (ATP, pink ; ADP, dark red ), ( c ) Same as panel b , viewed from the lower side and with subunit F and the VSL domains removed for clarity. ( d ) Type 1 binding pockets for alternating odd-numbered substrate amino acids, formed along the central channel of the Vps4 hexamer.…”