Structure and function of the human VPS4 proteins

To spread infection, HIV-1 must bud through the limiting membranes of producer cells. The HIV-1 genome does not encode the machinery necessary for viral egress, therefore, it must use host proteins for this purpose. HIV-1 Gag binds two proteins involved in multivesicular body (MVB) biogenesis, TSG101 and AIP1, and thus hijacks the proteins of the MVB pathway for virus release. TSG101 is one component of the complex ESCRT-I, which normally recruits ubiquitylated cargoes into the MVB pathway. ESCRT-I also recruits two other complexes to the site of vesicle formation, ESCRT-II and ESCRT-III. AIP1 interacts with ESCRT-I and ESCRT-III. The CHMP proteins of ESCRT-III bind the membrane and presumably build a protein lattice that mediates protein sorting and vesicle fission. VPS4, an AAA ATPase, binds directly to the CHMP proteins, uses the energy of ATP hydrolysis to disassemble ESCRT-III and releases them from the membrane. VPS4 mutants that cannot bind or hydrolyze ATP are dominant negative inhibitors of HIV-1 budding and cause arrest at a late stage in viral egress. This thesis focuses on understanding the structure and function of VPS4.; Chapter 2 presents the solution structure of the VPS4A MIT domain and demonstrates that this domain binds the C-terminal region of the ESCRT-III protein, CHMP1B. The crystal structure of monomeric VPS4B is reported in Chapter 3 along with biochemical analysis of the protein. The structure shows that VPS4 possesses the canonical AAA ATPase cassette, as well as two unique structural features: a three-stranded anti-parallel beta domain inserted within the ATPase domain and a novel C-terminal helix. VPS4 assembles into a ring-like oligomer containing 10--12 subunits upon ATP binding. There is conserved pore 1 motif that is located at the center of these rings. When this motif is mutated in VPS4A or VPS4B, HIV-1 budding is inhibited. We propose that VPS4B forms a double ring structure in which two hexameric rings form a double ring dodecamer and that VPS4 acts as a specialized chaperone that binds assembled ESCRT-III proteins and pulls them into its central pore, thereby releasing them from the membrane.