Bro1 domains are elongated, banana-shaped domains that were first identified in the yeast ESCRT pathway protein, Bro1p. Humans express three Bro1 domain-containing proteins: ALIX, BROX, and HD-PTP, which function in association with the ESCRT pathway to help mediate intraluminal vesicle formation at multivesicular bodies, the abscission stage of cytokinesis, and/or enveloped virus budding. Human Bro1 domains share the ability to bind the CHMP4 subset of ESCRT-III proteins, associate with the HIV-1 NC(Gag) protein, and stimulate the budding of viral Gag proteins. The curved Bro1 domain structure has also been proposed to mediate membrane bending. To date, crystal structures have only been available for the related Bro1 domains from the Bro1p and ALIX proteins, and structures of additional family members should therefore aid in the identification of key structural and functional elements.
The cellular ALIX protein functions within the ESCRT pathway to facilitate intralumenal endosomal vesicle formation, the abscission stage of cytokinesis, and enveloped virus budding. Here, we report that the C-terminal proline-rich region (PRR) of ALIX folds back against the upstream domains and auto-inhibits V domain binding to viral late domains. Mutations designed to destabilize the closed conformation of the V domain opened the V domain, increased ALIX membrane association, and enhanced virus budding. These observations support a model in which ALIX activation requires dissociation of the autoinhibitory PRR and opening of the V domain arms.
Retroviral Gag proteins contain short late-domain motifs that recruit cellular ESCRT pathway proteins to facilitate virus budding. ALIX-binding late domains often contain the core consensus sequence YPX(n)L (where X(n) can vary in sequence and length). However, some simian immunodeficiency virus (SIV) Gag proteins lack this consensus sequence, yet still bind ALIX. We mapped divergent, ALIX-binding late domains within the p6(Gag) proteins of SIV(mac239) ((40)SREKPYKEVTEDLLHLNSLF(59)) and SIV(agmTan-1) ((24)AAGAYDPARKLLEQYAKK(41)). Crystal structures revealed that anchoring tyrosines (in lightface) and nearby hydrophobic residues (underlined) contact the ALIX V domain, revealing how lentiviruses employ a diverse family of late-domain sequences to bind ALIX and promote virus budding.
HIV-1 and other enveloped viruses can be restricted by a host cellular protein called BST2/tetherin that prevents release of budded viruses from the cell surface. Mature BST2 contains a small cytosolic region, a predicted transmembrane helix, and an extracellular domain with a C-terminal GPI anchor. To advance understanding of BST2 function, we have determined a 2.6 ? crystal structure of the extracellular domain of the bacterially expressed recombinant human protein, residues 47-152, under reducing conditions. The structure forms a single long helix that associates as a parallel dimeric coiled coil over its C-terminal two-thirds, while the N-terminal third forms an antiparallel four-helix bundle with another dimer, creating a global tetramer. We also report the 3.45 ? resolution structure of BST2(51-151) prepared by expression as a secreted protein in HEK293T cells. This oxidized construct forms a dimer in the crystal that is superimposable with the reduced protein over the C-terminal two-thirds of the molecule, and its N terminus suggests pronounced flexibility. Hydrodynamic data demonstrated that BST2 formed a stable tetramer under reducing conditions and a dimer when oxidized to form disulfide bonds. A mutation that selectively disrupted the tetramer (L70D) increased protein expression modestly but only reduced antiviral activity by approximately threefold. Our data raise the possibility that BST2 may function as a tetramer at some stage, such as during trafficking, and strongly support a model in which the primary functional state of BST2 is a parallel disulfide-bound coiled coil that displays flexibility toward its N terminus.
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