Crystallization and Biophysical Approaches for Studying the Interactions Between the Vps4-MIT Domain and ESCRT-III Proteins.

Abstract

The AAA ATPase Vps4 disassembles the ESCRT complex from the endosomal membrane. Vps4 contains an N-terminal MIT (microtubule interacting and transport) domain and a C-terminal catalytic domain. The MIT domain binds to MIMs (MIT-interacting motifs), which exist at the C-terminus of ESCRT-III proteins, with a dissociation constant in the micromolar range. Five MIMs have been identified by structural and biophysical methods to date, and the recognition motifs have been refined. Among biophysical approaches used to analyze protein interactions, surface plasmon resonance (SPR) analysis is often suitable for weak interactions, and fluorescence-binding assay has an advantage in terms of sensitivity. We have introduced protein modification tags into the N-terminus of proteins with bacterial expression vectors for biotinylation and FlAsH (fluorescein arsenical hairpin binder) fluorescent labeling. Here, we describe how to purify the MIT domain of Vps4 and the MIMs of ESCRT-III proteins and how to conduct crystallography, SPR, and fluorescence-binding assays.