Background: Vps4 ATPase is stimulated by the interaction between Vta1 and Vps60, but the structural basis for this interaction remains unclear. Results: The structure of the Vta1 N-terminal domain (Vta1NTD) in complex with Vps60(128 -186) was determined. Conclusion: Vps60(128 -186) interacts with Vta1NTD through helices ␣4Ј and ␣5Ј, extending over Vta1NTD MIT2 domain helices 1-3. Significance: This is a novel MIT recognition mode.

Background: LIP5 and ESCRT-III are regulators of VPS4 in biological processes that require the ESCRT function. Results: Structural and functional analyses of human VPS4, LIP5, and ESCRT-III interactions are presented. Conclusion: ESCRT-III protein CHMP5 inhibits LIP5-mediated VPS4 activation by inducing a moderate conformational change within LIP5. Significance: This study reveals important mechanistic differences in VPS4 regulation between fungi and metazoans.

Background: Vfa1 was recently identified as a Vps4-binding protein, yet its function was unknown. Results: Vfa1 potently stimulates Vps4 ATPase activity, and Vps4-Vfa1 interaction consists of a binding mechanism previously seen in other Vps4 regulator structures. Conclusion: Biochemical and biophysical studies show that Vfa1 is a novel regulator of Vps4 in the multivesicular body pathway. Significance: Vps4-Vfa1 interaction further expands the complexity of Vps4 regulation.

Vta1 and Vps60 are two ESCRT associated proteins, their direct interaction enhances Vps4 ATPase activity. The N-terminal domain of Vta1 (residues 1–167aa, named as Vta1NTD) contains two tandem MIT domains, which specifically recognize Vps60 and Did2 but not other ESCRT-III subunits. The fragment Vps60 (128–186aa) was reported to display full activity of Vps60, which stimulates Vps4 ATPase in a Vta1-dependent manner. To study the structural basis for the interaction between Vta1 and Vps60, as a first step, here, we report the resonance assignments of the sequential backbone atoms and the side chains of the residues in the two components of Vta1NTD/Vps60128–186 complex at pH 7.0 and 20 °C (BMRB No. 18521).

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