Structural and mechanistic studies of VPS4 proteins.
VPS4 ATPases function in multivesicular body formation and in HIV-1 budding. Here, we report the crystal structure of monomeric apo human VPS4B/SKD1 (hVPS4B), which is composed of five distinct elements: a poorly ordered N-terminal MIT domain that binds ESCRT-III substrates, large (mixed alpha/beta) and small (alpha) AAA ATPase domains that closely resemble analogous domains in the p97 D1 ATPase cassette, a three-stranded antiparallel beta domain inserted within the small ATPase domain, and a novel C-terminal helix. Apo hVPS4B and yeast Vps4p (yVps4p) proteins dimerized in solution, and assembled into larger complexes (10-12 subunits) upon ATP binding. Human and yeast adaptor proteins (LIP5 and yVta1p, respectively) bound the beta domains of the fully assembled hVPS4B and yVps4p proteins. We therefore propose that Vps4 proteins cycle between soluble, inactive low molecular weight complexes and active, membrane-associated double-ring structures that bind ATP and coassemble with LIP5/Vta1. Finally, HIV-1 budding was inhibited by mutations in a loop that projects into the center of the modeled hVPS4B rings, suggesting that hVPS4B may release the assembled ESCRT machinery by pulling ESCRT-III substrates up into the central pore.
Scott
Anna
A
Department of Biochemistry, University of Utah, Salt Lake City, UT 84132-3201, USA.
Chung
Hyo-Young
HY
Gonciarz-Swiatek
Malgorzata
M
Hill
Gina C
GC
Whitby
Frank G
FG
Gaspar
Jason
J
Holton
James M
JM
Viswanathan
Ramya
R
Ghaffarian
Sanaz
S
Hill
Christopher P
CP
Sundquist
Wesley I
WI
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry; D003461:Crystallography; D056827:Endosomal Sorting Complexes Required for Transport; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D033921:Vesicular Transport Proteins / Q000737:chemistry
D002352:Carrier Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C487285:VTA1 protein, S cerevisiae; C499837:VTA1 protein, human; D033921:Vesicular Transport Proteins; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; C490304:VPS4B protein, human