ALIX/AIP1 functions in enveloped virus budding, endosomal protein sorting, and many other cellular processes. Retroviruses, including HIV-1, SIV, and EIAV, bind and recruit ALIX through YPX n L late-domain motifs (X = any residue; n = 1-3). Crystal structures reveal that human ALIX is composed of an N-terminal Bro1 domain and a central domain that is composed of two extended three-helix bundles that form elongated arms that fold back into a ''V.'' The structures also reveal conformational flexibility in the arms that suggests that the V domain may act as a flexible hinge in response to ligand binding. YPX n L late domains bind in a conserved hydrophobic pocket on the second arm near the apex of the V, whereas CHMP4/ ESCRT-III proteins bind a conserved hydrophobic patch on the Bro1 domain, and both interactions are required for virus budding. ALIX therefore serves as a flexible, extended scaffold that connects retroviral Gag proteins to ESCRT-III and other cellular-budding machinery.

Bispecific antibodies are on the cusp of coming of age as therapeutics more than half a century after they were first described. Two bispecific antibodies, catumaxomab (Removab(®), anti-EpCAM×anti-CD3) and blinatumomab (Blincyto(®), anti-CD19×anti-CD3) are approved for therapy, and >30 additional bispecific antibodies are currently in clinical development. Many of these investigational bispecific antibody drugs are designed to retarget T cells to kill tumor cells, whereas most others are intended to interact with two different disease mediators such as cell surface receptors, soluble ligands and other proteins. The modular architecture of antibodies has been exploited to create more than 60 different bispecific antibody formats. These formats vary in many ways including their molecular weight, number of antigen-binding sites, spatial relationship between different binding sites, valency for each antigen, ability to support secondary immune functions and pharmacokinetic half-life. These diverse formats provide great opportunity to tailor the design of bispecific antibodies to match the proposed mechanisms of action and the intended clinical application.

Retrovirus budding requires short peptide motifs (late domains) located within the viral Gag protein that function by recruiting cellular factors. The YPX(n)L late domains of HIV and other lentiviruses recruit the protein ALIX (also known as AIP1), which also functions in vesicle formation at the multivesicular body and in the abscission stage of cytokinesis. Here, we report the crystal structures of ALIX in complex with the YPX(n)L late domains from HIV-1 and EIAV. The two distinct late domains bind at the same site on the ALIX V domain but adopt different conformations that allow them to make equivalent contacts. Binding studies and functional assays verified the importance of key interface residues and revealed that binding affinities are tuned by context-dependent effects. These results reveal how YPX(n)L late domains recruit ALIX to facilitate virus budding and how ALIX can bind YPX(n)L sequences with both n = 1 and n = 3.

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 Å 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 Å 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.coiled coil | crystal structures | HIV | innate immunity | restriction factor

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 polyproteins contain short sequence motifs, termed "late domains," that facilitate virus budding by recruiting components of the cellular ESCRT pathway (4, 38). For example, the HIV-1 p6Gag protein contains "PTAP" and "YPXL" late domains (designated by their consensus sequences), that bind directly to the TSG101 and ALIX proteins, respectively (6,9,19,33,39). ALIX, in turn, binds the CHMP4 subunits of the ESCRT-III complex, resulting in recruitment of the VPS4 ATPase, membrane fission, and virus release (10, 27).ALIX contains three distinct structural elements: an N-terminal Bro1 domain, a central V domain, and a C-terminal proline-rich region (PRR). The boomerang-shaped Bro1 domain binds CHMP4 proteins (7,13,20), the V domain comprises two extended three-helix bundles and binds YPXL late domains (7,16,44,45), and the PRR binds a series of other proteins but is predicted to lack a persistent secondary or tertiary structure (7,8,24). Like other ESCRT factors, ALIX must cycle between soluble (inactive) and membrane-associated (active) states. Several lines of evidence suggest that conformational changes accompany (or induce) these transitions. First, recombinant ALIX proteins can form stable monomers and dimers (7, 23), and biochemical evidence suggests that the dimer is the active conformation (5,7,23,29). Second, smallangle X-ray scattering (SAXS) profiles indicate that the two arms of the V domain may open and associate in an antiparallel fashion when the protein dimerizes (29). Third, recent reports show that the PRR can inhibit ALIX binding to conformationally sensitive monoclonal antibodies, CHMP4 proteins, and viral Gag proteins (46-48). However, previous studies have not characterized the structure or conformational transitions of pure, full-length ALIX, because this protein has not been available.Although we were unable to express full-length human ALIX protein in Escherichia coli, we could produce multimilligram quantities of pure recombinant ALIX in insect cells using a baculoviral expression system. Briefly, 2 liters of SF21 cells were infected with a BaculoDirect (Invitrogen) expression vector, which encoded His 6 -ALIX (ALIX residues 1 to 868, WISP10-643). The cells were lysed by sonication 48 h postinfection (300 mM NaCl, 10 mM imidazole, 5% [vol/vol] (Fig. 1A). This procedure typically yielded 5 mg of pure monomeric ALIX, and the protein ide...