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Like most enveloped viruses, HIV must acquire a lipid membrane as it assembles and buds through the plasma membrane of infected cells to spread infection. Several sets of host cell machinery facilitate this process, including proteins of the endosomal sorting complexes required for transport pathway, which mediates the membrane fission reaction required to complete viral budding, as well as angiomotin (AMOT) and NEDD4L, which bind one another and promote virion membrane envelopment. AMOT and NEDD4L interact through the four NEDD4L WW domains and three different AMOT Pro-Pro-x (any amino acid)-Tyr (PPxY) motifs, but these interactions are not yet well defined. Here, we report that individual AMOT PPxY and NEDD4L WW domains interact with the following general affinity hierarchies: AMOT PPxY1>PPxY2>PPxY3 and NEDD4L WW3>WW2>WW1∼WW4. The unusually high-affinity of the AMOT PPxY1–NEDD4L WW3 interaction accounts for most of the AMOT–NEDD4L binding and is critical for stimulating HIV-1 release. Comparative structural, binding, and virological analyses reveal that complementary ionic and hydrophobic contacts on both sides of the WW–PPxY core interaction account for the unusually high affinity of the AMOT PPxY1–NEDD4L WW3 interaction. Taken together, our studies reveal how the first AMOT PPxY1 motif binds the third NEDD4L WW domain to stimulate HIV-1 viral envelopment and promote infectivity.

Cytokinetic abscission facilitates the irreversible separation of daughter cells. This process requires the endosomal-sorting complexes required for transport (ESCRT) machinery and is tightly regulated by charged multivesicular body protein 4C (CHMP4C), an ESCRT-III subunit that engages the abscission checkpoint (NoCut) in response to mitotic problems such as persisting chromatin bridges within the midbody. Importantly, a human polymorphism in CHMP4C (rs35094336, CHMP4C T232 ) increases cancer susceptibility. Here, we explain the structural and functional basis for this cancer association: The CHMP4C T232 allele unwinds the C-terminal helix of CHMP4C, impairs binding to the early-acting ESCRT factor ALIX, and disrupts the abscission checkpoint. Cells expressing CHMP4C T232 exhibit increased levels of DNA damage and are sensitized to several conditions that increase chromosome missegregation, including DNA replication stress, inhibition of the mitotic checkpoint, and loss of p53. Our data demonstrate the biological importance of the abscission checkpoint and suggest that dysregulation of abscission by CHMP4C T232 may synergize with oncogene-induced mitotic stress to promote genomic instability and tumorigenesis.

Cytokinetic abscission facilitates the irreversible separation of daughter cells. This process requires the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery and is tightly regulated by Charged Multivesicular body Protein 4C (CHMP4C), an ESCRT-III subunit that engages the abscission checkpoint (NoCut) in response to mitotic problems such as persisting chromatin bridges within the midbody. Importantly, a human polymorphism in CHMP4CT232 (rs35094336), increases cancer susceptibility. Here, we explain the structural and functional basis for this cancer association: the CHMP4CT232 allele unwinds the C-terminal helix of CHMP4C, impairs binding to the early-acting ESCRT factor ALIX, and disrupts the abscission checkpoint. Cells expressing CHMP4CT232 exhibit increased levels of DNA damage and are sensitized to several conditions that increase chromosome mis-segregation, including DNA replication stress, inhibition of the mitotic checkpoint, and loss of p53. Our data demonstrate the biological importance of the abscission checkpoint, and suggest that dysregulation of abscission by CHMP4CT232 may synergize with oncogene-induced mitotic stress to promote genomic instability and tumorigenesis.Significance StatementThe final step of cell division, abscission, is temporally regulated by the Aurora B kinase and CHMP4C in a conserved pathway called the abscission checkpoint which arrests abscission in the presence of lingering mitotic problems. Despite extensive study, the physiological importance of this pathway to human health has remained elusive. We now demonstrate that a cancer predisposing polymorphism in CHMP4C disrupts the abscission checkpoint and results in DNA damage accumulation. Moreover, deficits in this checkpoint synergize with p53 loss and generate aneuploidy under stress conditions that increase the frequency of chromosome missegregation. Therefore, cells expressing the cancer-associated polymorphism in CHMP4C are genetically unstable, thus suggesting a novel oncogenic mechanism that may involve the dysregulation of abscission.

Restriction factors and pattern recognition receptors are important components of intrinsic cellular defenses against viral infection. Mammalian TRIM5α proteins are restriction factors and receptors that target the capsid cores of retroviruses and activate ubiquitin-dependent antiviral responses upon capsid recognition. Here, we report crystallographic and functional studies of the TRIM5α B-box 2 domain, which mediates higher-order assembly of TRIM5 proteins. The B-box can form both dimers and trimers, and the trimers can link multiple TRIM5α proteins into a hexagonal net that matches the lattice arrangement of capsid subunits and enables avid capsid binding. Two modes of conformational flexibility allow TRIM5α to accommodate the variable curvature of retroviral capsids. B-box mediated interactions also modulate TRIM5α’s E3 ubiquitin ligase activity, by stereochemically restricting how the N-terminal RING domain can dimerize. Overall, these studies define important molecular details of cellular recognition of retroviruses, and how recognition links to downstream processes to disable the virus. DOI: http://dx.doi.org/10.7554/eLife.16309.001, eLife digest After infecting a cell, a virus reprograms the cell to produce new copies of the virus, which then spread to other cells. However, cells have evolved ways to fight back against this infection. For example, many mammalian cells contain proteins called restriction factors that prevent the virus from multiplying. The TRIM5 proteins form one common set of restriction factors that act against a class of viruses called retroviruses. HIV-1 and related retroviruses have a protein shell known as a capsid that surrounds the genetic material of the virus. The capsid contains several hundred repeating units, each of which consists of a hexagonal ring of six capsid proteins. Although this basic pattern is maintained across different retroviruses, the overall shape of the capsids can vary considerably. For instance, HIV-1 capsids are shaped like a cone, but other retroviruses can form cylinders or spheres. Soon after a retrovirus enters a mammalian cell, TRIM5 proteins bind to the capsid. This causes the capsid to be destroyed, which prevents replication of the virus. Previous research has shown that many TRIM5 proteins must link up with each other via a region of their structure called the 'B-box 2' domain in order to efficiently recognize capsids. How this assembly process occurs, and why it enables the TRIM5 proteins to recognize different capsids was not fully understood. Now, Wagner et al. (and independently Li, Chandrasekaran et al.) have investigated these questions. Wagner et al. engineered short versions of a type of TRIM5 protein called TRIM5α and used a technique called X-ray crystallography to determine the structure of its B-box domain. This revealed that the B-box present in one molecule of TRIM5α can associate with the B-boxes on two other TRIM5α molecules. By working in groups of three (or trimers), the B-box domains connect several TRIM5α proteins to form a hexagonal net. The TRIM5α net matches the arrangement of the capsid proteins in the shell of the virus, which enables TRIM5α to bind strongly to HIV-1 capsids. Wagner et al. also found that B-box trimers are flexible, which allows the TRIM5α net to adapt to the shape of the HIV-1 capsid and wrap around regions where it curves. In addition, computer modelling suggested that the B-box trimer may also enable TRIM5α to carry out the next steps in the process of disabling the virus. Further work is now needed to understand in more detail how the trimers have this effect. DOI: http://dx.doi.org/10.7554/eLife.16309.002

The nuclear pore complex is the gateway for selective traffic between the nucleus and cytoplasm. To learn how building blocks of the pore can create specific docking sites for transport receptors and regulatory factors, we have studied a zinc finger module present in multiple copies within the nuclear pores of higher eukaryotes. All four zinc fingers of human Nup153 were found to bind the small GTPase Ran with dissociation constants ranging between 5 and 40 mum. In addition a fragment of Nup153 encompassing the four tandem zinc fingers was found to bind Ran with similar affinity. NMR structural studies revealed that a representative Nup153 zinc finger adopts the same zinc ribbon structure as the previously characterized Npl4 NZF module. Ran binding was mediated by a three-amino acid motif (Leu(13)/Val(14)/Asn(25)) located within the two zinc coordination loops. Nup153 ZnFs bound GDP and GTP forms of Ran with similar affinities, indicating that this interaction is not influenced by a nucleotide-dependent conformational switch. Taken together, these studies elucidate the Ran-binding interface on Nup153 and, more broadly, provide insight into the versatility of this zinc finger binding module.

The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations.

The Rybp protein has been promoted as a Polycomb group (PcG)-associated protein, but its molecular function has remained elusive. Here we show that Rybp is a novel ubiquitin binding protein and is itself ubiquitinated. The Rybp interacting PcG protein Ring1B, a known ubiquitin E3 ligase, promotes Rybp ubiquitination. Moreover, one target of Rybp’s ubiquitin binding domain appears to be ubiquitinated histone H2A; this histone is a substrate for Ring1B’s E3 ligase activity in association with gene silencing processes. These findings on Rybp provide a further link between the ubiquitination system and PcG transcriptional repressors.

Restriction factors are important components of intrinsic cellular defense mechanisms against viral pathogens. TRIM5α is a restriction factor that intercepts the incoming capsid cores of retroviruses such as HIV and provides an effective species-specific barrier to retroviral infection. The TRIM5α SPRY domain directly binds the capsid with only very weak, millimolar-level affinity, and productive capsid recognition therefore requires both TRIM5α dimerization and assembly of the dimers into a multivalent hexagonal lattice to promote avid binding. Here, we explore the important unresolved question of whether the SPRY domains are flexibly linked to the TRIM lattice or more precisely positioned to maximize avidity. Biochemical and biophysical experiments indicate that the linker segment connecting the SPRY domain to the coiled-coil domain adopts an α-helical fold, and that this helical portion mediates interactions between the two domains. Targeted mutations were generated to disrupt the putative packing interface without affecting dimerization or higher-order assembly, and we identified mutant proteins that were nevertheless deficient in capsid binding in vitro and restriction activity in cells. Our studies therefore support a model wherein substantial avidity gains during assembly-mediated capsid recognition by TRIM5α come in part from tailored spacing of tethered recognition domains., Author summary TRIM5α is a cytosolic protein that provides effective protection for mammalian cells against retroviral infection. This anti-viral defense mechanism is an unprecedented example of how the cell can recognize entire capsids, which are large, megadalton-sized particles. TRIM5α achieves this by assembling into a hexagonal scaffold that coats the capsid. An important unresolved question is how the capsid-binding SPRY domain of TRIM5α is positioned to optimize its contact points on the capsid surface. Here, we use a variety of techniques to determine that the SPRY domains in the TRIM lattice are organized in pairs and likely to be stably tethered against the hexagonal scaffold. Such an arrangement maximizes the avidity of capsid binding, and allows TRIM5α to act as a “molecular ruler” that matches the spacings and orientations of the capsid subunits.

Native-state amide hydrogen exchange (HX) of proteins in the presence of denaturant has provided valuable details on the structures of equilibrium folding intermediates. Here, we extend HX theory to model thiol group exchange (SX) in single cysteine-containing variants of sperm whale ferric aquomyoglobin. SX is complementary to HX in that it monitors conformational opening events that expose side-chains, rather than the main chain, to solvent. A simple two-process model, consisting of EX2-limited local structural fluctuations and EX1-limited global unfolding, adequately accounts for all HX data. SX is described by the same model except at very low denaturant concentrations and when the bulky labeling reagent 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) is used. Under these conditions SX can occur by a novel denaturant-dependent process. This anomalous behavior is not observed when the smaller labeling reagent methyl methanethiosulfonate is employed, suggesting that it reflects a denaturant-induced increase in the amplitudes of local structural fluctuations. It also is not seen in heme-free apomyoglobin, which may indicate that local openings are sufficiently large in the absence of denaturant to allow DTNB unhindered access. Differences in SX kinetics obtained using the two labeling reagents provide estimates of the sizes of local opening reactions at different sites in the protein. At all sequence positions examined except for position 73, the same opening event appears to facilitate exchange of both backbone amide and side-chain thiol groups. The C73 thiol group is exposed by a low-energy fluctuation that does not expose its amide group to exchange.

The globin derived from the monomer Component IV hemoglobin of the marine amnelid, Glycera dibranchiata, has been completely sequenced, and the resulting information has been used to create a structural model of the protein. The most important result is that the consensus sequence of Component IV differs by 3 amino acids from a cDNA-predicted amino acid sequence thought earlier to encode the Component IV hemoglobin. This work reveals that the histidine (E7), typical of most heme-containing globins, is replaced by leucine in Component IV. Also significant is that this sequence is not identical to any of the previously reported Glycera dibranchiata monomer hemoglobin sequences, including the sequence from a previously reported crystal structure, but has high identity to all. A three-dimensional structural model for monomer Component IV hemoglobin was constructed using the published 1.5 A crystal structure of a monomer hemoglobin from Glycera dibranchiata as a template. The model shows several interesting features: (1) a Phe31 (B10) that is positioned in the active site; (2) a His39 occurs in an interhelical region occupied by Pro in 98.2% of reported globin sequences; and (3) a Met41 is found at a position that emerges from this work as a previously unrecognized heme contact.

The rhesus monkey intrinsic immunity factor TRIM5α rh recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV), and inhibits the accumulation of viral reverse transcripts. However, direct interactions between restricting TRIM5α proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. To facilitate structural and mechanistic studies of retroviral restriction, we have developed methods for expressing and purifying an active chimeric TRIM5α rh protein containing the RING domain from the related human TRIM21 protein. This recombinant TRIM5-21R protein was expressed in SF-21 insect cells and purified through three chromatographic steps. Two distinct TRIM5-21R species were purified and shown to correspond to monomers and dimers, as analyzed by analytical ultracentrifugation. Chemically cross-linked recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5α proteins exhibited similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities, indicating that mammalian TRIM5α proteins are predominantly dimeric. Purified TRIM5-21R had ubiquitin ligase activity and could autoubquitylate with different E2 ubiquitin conjugating enzymes in vitro. TRIM5-21R bound directly to synthetic capsids composed of recombinant HIV-1 CA-NC proteins and to authentic EIAV core particles. HIV-1 CA-NC assemblies bound dimeric TRIM5-21R better than either monomeric TRIM5-21R or TRIM5-21R constructs that lacked the SPRY domain or its V1 loop. Thus, our studies indicate that TRIM5α proteins are dimeric ubiquitin E3 ligases that recognize retroviral capsids through direct interactions mediated by the SPRY domain and demonstrate that these activities can be recapitulated in vitro using pure recombinant proteins.

The ESCRT-I and ESCRT-II complexes help sort ubiquitinated proteins into vesicles that accumulate within multivesicular bodies (MVBs). Crystallographic and biochemical analyses reveal that the GLUE domain of the human ESCRT-II EAP45 (also called VPS36) subunit is a split pleckstrin-homology domain that binds ubiquitin along one edge of the beta-sandwich. The structure suggests how human ESCRT-II can couple recognition of ubiquitinated cargoes and endosomal phospholipids during MVB protein sorting.

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 m icrotubule i nteracting and t ransport (MIT) domain and demonstrate that the VPS4A MIT domain binds the C-terminal half of the ESCRT-III protein, CHMP1B ( K d = 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.

The mitochondrial protein frataxin is essential for cellular regulation of iron homeostasis. Although the exact function of frataxin is not yet clear, recent reports indicate the protein binds iron and can act as a mitochondrial iron chaperone to transport Fe(II) to ferrochelatase and ISU proteins within the heme and iron-sulfur cluster biosynthetic pathways, respectively. We have determined the solution structure of apo yeast frataxin to provide a structural basis of how frataxin binds and donates iron to the ferrochelatase. While the protein's alpha-beta-sandwich structural motif is similar to that observed for human and bacterial frataxins, the yeast structure presented in this report includes the full N-terminus observed for the mature processed protein found within the mitochondrion. In addition, NMR spectroscopy was used to identify frataxin amino acids that are perturbed by the presence of iron. Conserved acidic residues in the helix 1-strand 1 protein region undergo amide chemical shift changes in the presence of Fe(II), indicating a possible iron-binding site on frataxin. NMR spectroscopy was further used to identify the intermolecular binding interface between ferrochelatase and frataxin. Ferrochelatase appears to bind to frataxin's helical plane in a manner that includes its iron-binding interface.

Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains. One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes. We now report the solution structure of the NZF domain from Npl4 in complex with Ub. The structure reveals that three key NZF residues (13TF14/M25) surrounding the zinc coordination site bind the hydrophobic 'Ile44' surface of Ub. Mutations in the 13TF14/M25 motif inhibit Ub binding, and naturally occurring NZF domains that lack the motif do not bind Ub. However, substitution of the 13TF14/M25 motif into the nonbinding NZF domain from RanBP2 creates Ub-binding activity, demonstrating the versatility of the NZF scaffold. Finally, NZF mutations that inhibit Ub binding by the NZF domain of Vps36/ESCRT-II also inhibit sorting of ubiquitylated proteins into the yeast vacuole. Thus, the NZF is a versatile protein recognition domain that is used to bind ubiquitylated proteins during vacuolar protein sorting, and probably many other biological processes.

Ubiquitylated proteins are directed into a large number of different cellular pathways through interactions with effector proteins that contain conserved ubiquitin binding motifs. Here, we report the solution structure and ubiquitin binding properties of one such motif, the Npl4 zinc finger or RanBP2/Nup358 zinc finger (NZF) domain. Npl4 NZF forms a compact module composed of four antiparallel beta-strands linked by three ordered loops. A single zinc ion is coordinated by four conserved cysteines from the first and third loops, which form two rubredoxin knuckles. Npl4 NZF binds specifically, but weakly, to free ubiquitin using a conserved 13TF14 dipeptide to interact with the "Ile-44" surface of ubiquitin. Our studies reveal the structure of this versatile class of protein binding domains and provide a means for identifying the subset of NZF domains likely to bind ubiquitin.

The structural proteins of HIV and Ebola display PTAP peptide motifs (termed 'late domains') that recruit the human protein Tsg101 to facilitate virus budding. Here we present the solution structure of the UEV (ubiquitin E2 variant) binding domain of Tsg101 in complex with a PTAP peptide that spans the late domain of HIV-1 p6(Gag). The UEV domain of Tsg101 resembles E2 ubiquitin-conjugating enzymes, and the PTAP peptide binds in a bifurcated groove above the vestigial enzyme active site. Each PTAP residue makes important contacts, and the Ala 9-Pro 10 dipeptide binds in a deep pocket of the UEV domain that resembles the X-Pro binding pockets of SH3 and WW domains. The structure reveals the molecular basis of HIV PTAP late domain function and represents an attractive starting point for the design of novel inhibitors of virus budding.

Human Tsg101 plays key roles in HIV budding and in cellular vacuolar protein sorting (VPS). In performing these functions, Tsg101 binds both ubiquitin (Ub) and the PTAP tetrapeptide 'late domain' motif located within the viral Gag protein. These interactions are mediated by the N-terminal domain of Tsg101, which belongs to the catalytically inactive ubiquitin E2 variant (UEV) family. We now report the structure of Tsg101 UEV and chemical shift mapping of the Ub and PTAP binding sites. Tsg101 UEV resembles canonical E2 ubiquitin conjugating enzymes, but has an additional N-terminal helix, an extended beta-hairpin that links strands 1 and 2, and lacks the two C-terminal helices normally found in E2 enzymes. PTAP-containing peptides bind in a hydrophobic cleft exposed by the absence of the C-terminal helices, whereas ubiquitin binds in a novel site surrounding the beta-hairpin. These studies provide a structural framework for understanding how Tsg101 mediates the protein-protein interactions required for HIV budding and VPS.

The sorting and degradation of cell-surface proteins are essential for cellular homeostasis. The ESCRT-I complex is known to be involved in these events, and new structural findings elucidate its core architecture.