Disassembly of the endosomal sorting complex required for transport (ESCRT) 
machinery from biological membranes is a critical final step in cellular 
processes that require the ESCRT function. This reaction is catalyzed by VPS4, 
an AAA-ATPase whose activity is tightly regulated by a host of proteins, 
including LIP5 and the ESCRT-III proteins. Here, we present structural and 
functional analyses of molecular interactions between human VPS4, LIP5, and the 
ESCRT-III proteins. The N-terminal domain of LIP5 (LIP5NTD) is required for 
LIP5-mediated stimulation of VPS4, and the ESCRT-III protein CHMP5 strongly 
inhibits the stimulation. Both of these observations are distinct from what was 
previously described for homologous yeast proteins. The crystal structure of 
LIP5NTD in complex with the MIT (microtubule-interacting and 
transport)-interacting motifs of CHMP5 and a second ESCRT-III protein, CHMP1B, 
was determined at 1 Å resolution. It reveals an ESCRT-III binding induced 
moderate conformational change in LIP5NTD, which results from insertion of a 
conserved CHMP5 tyrosine residue (Tyr(182)) at the core of LIP5NTD structure. 
Mutation of Tyr(182) partially relieves the inhibition displayed by CHMP5. 
Together, these results suggest a novel mechanism of VPS4 regulation in 
metazoans, where CHMP5 functions as a negative allosteric switch to control 
LIP5-mediated stimulation of VPS4.