Structure and ESCRT-III Protein Interactions of the MIT Domain of Human VPS4A

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 102; no. 39; pp. 13813 - 13818
• Main Authors: Scott, Anna, Gaspar, Jason, Stuchell-Brereton, Melissa D., Alam, Steven L., Skalicky, Jack J., Sundquist, Wesley I., Coffin, John M.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 27.09.2005; National Acad Sciences
• Subjects: Adenosine triphosphatase; Adenosine triphosphatases; Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Atoms; ATPases Associated with Diverse Cellular Activities; Biochemistry; Biological Sciences; Biosensing techniques; Budding; Chemical equilibrium; Conserved Sequence; DNA; DNA Mutational Analysis; Endosomal Sorting Complexes Required for Transport; HIV; Human immunodeficiency virus; Human immunodeficiency virus 1; Humans; Leucine - genetics; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Microtubules - metabolism; Molecular Sequence Data; Mutation; NMR; Nuclear magnetic resonance; Nuclear Proteins - chemistry; Nuclear Proteins - metabolism; Protein Interaction Mapping; Protein Structure, Secondary; Proteins; Repressor Proteins - chemistry; Repressor Proteins - genetics; Repressor Proteins - metabolism; Social interaction; Vacuolar Proton-Translocating ATPases; Vesicular Transport Proteins; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0502165102

The VPS4 AAA ATPases function both in endosomal vesicle formation and in the budding of many enveloped RNA viruses, including HIV-1, VPS4 proteins act by binding and catalyzing release of the membrane-associated ESCRT-III protein lattice, thereby allowing multiple rounds of protein sorting and vesicle formation. Here, we report the solution structure of the N-terminal VPS4A$\underline{\text{m}}\text{icrotubule}$ $\underline{\text{i}}\text{nteracting}$and$\underline{\text{t}}\text{ransport}$(MIT) domain and demonstrate that the VPS4A MIT domain binds the C-terminal half of the ESCRT-III protein, CHMP1B (Kd=20± 13 μM). The MIT domain forms an asymmetric three-helix bundle that resembles the first three helices in a tetratricopeptide repeat (TPR) motif. Unusual interhelical interactions are mediated by a series of conserved aromatic residues that form coiled-coil interactions between the second two helices and also pack against the conserved alanines that interdigitate between the first two helices. Mutational analyses revealed that a conserved leucine residue (Leu-64) on the third helix that would normally bind the fourth helix in an extended TPR is used to bind CHMP1B, raising the possibility that ESCRT-III proteins may bind by completing the TPR motif.