Search

Human cytomegalovirus (CMV) is a ubiquitously distributed pathogen whose rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted global proteome profiling of rat CMV-infected cells and uncovered a pronounced loss of the transcription factor STAT2, which is crucial for antiviral interferon signalling. Via deletion mutagenesis, we found that the viral protein E27 is required for CMV-induced STAT2 depletion. Cellular and in vitro analyses showed that E27 exploits host-cell Cullin4-RING ubiquitin ligase (CRL4) complexes to induce poly-ubiquitylation and proteasomal degradation of STAT2. Cryo-electron microscopy revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DCAFs (DDB1- and Cullin4-associated factors), thereby displacing them from the catalytic core of CRL4. Moreover, structural analyses showed that E27 recruits STAT2 through a bipartite binding interface, which partially overlaps with the IRF9 binding site. Structure-based mutations in M27, the murine CMV homologue of E27, impair the interferon-suppressing capacity and virus replication in mouse models, supporting the conserved importance of DCAF mimicry for CMV immune evasion.

The SARS-CoV2 Omicron variant sub-lineages spread rapidly through the world, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for anti-SARS-CoV-2 agents that are effective against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production and potential for delivery via inhalation. Here, we characterize the RBD-specific nanobody W25, which we previously isolated from an alpaca, and show superior neutralization activity towards Omicron lineage BA.1 in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike surface glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. Furthermore, we show that W25 also binds the spike protein from the emerging, more infectious Omicron BA.2 lineage with picomolar affinity.In vivoevaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse prioritization of W25 for further clinical development.

Targeted protein degradation induced by heterobifunctional compounds and molecular glues presents an exciting avenue for chemical probe and drug discovery. To date, small-molecule ligands have been discovered for only a limited number of E3 ligases, which is an important limiting factor for realizing the full potential of targeted protein degradation. We report herein the discovery by chemical proteomics of azetidine acrylamides that stereoselectively and site-specifically react with a cysteine (C1113) in the E3 ligase substrate receptor DCAF1. We demonstrate that the azetidine acrylamide ligands for DCAF1 can be developed into electrophilic proteolysis-targeting chimeras (PROTACs) that mediated targeted protein degradation in human cells. We show that this process is stereoselective and does not occur in cells expressing a C1113A mutant of DCAF1. Mechanistic studies indicate that only low fractional engagement of DCAF1 is required to support protein degradation by electrophilic PROTACs. These findings, taken together, demonstrate how the chemical proteomic analysis of stereochemically defined electrophilic compound sets can uncover ligandable sites on E3 ligases that support targeted protein degradation.

Human cytomegalovirus (CMV) is a highly relevant and ubiquitously distributed human pathogen. Its rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted the first global proteome profiling of rat CMV-infected cells and uncovered a pronounced loss of the transcription factor STAT2, which is crucial for interferon signalling. Deletion mutagenesis documented that STAT2 is targeted by the viral protein E27. Cellular and in vitro analyses showed that E27 exploits host-derived Cullin4-RING ubiquitin ligases (CRL4) to induce poly-ubiquitylation and proteasomal degradation of STAT2. A cryo-electron microscopic structure determination revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DDB1- and Cullin4-associated factors (DCAFs) to displace them from the catalytic core of CRL4. Moreover, structural analyses elucidated the mechanism of STAT2 recruitment and indicate that E27-binding additionally disturbs STAT2-dependent interferon signalling by occupying its IRF9 binding interface. For the first time, these data provide structural insights into cytomegalovirus-encoded interferon antagonism and establish an atomic model for STAT2 counteraction by CRL4 misappropriation with important implications for viral immune evasion.

The biological role of RNA-binding proteins in the secretory pathway is not well established. Here, we describe that human HDLBP/Vigilin directly interacts with more than 80% of ER-localized mRNAs. PAR-CLIP analysis reveals that these transcripts represent high affinity HDLBP substrates and are specifically bound in their coding sequences (CDS), in contrast to CDS/3’UTR-bound cytosolic mRNAs. HDLBP crosslinks strongly to long CU-rich motifs, which frequently reside in CDS of ER-localized mRNAs and result in high affinity multivalent interactions. In addition to HDLBP-ncRNA interactome, quantification of HDLBP-proximal proteome confirms association with components of the translational apparatus and the signal recognition particle. Absence of HDLBP results in decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in model cell lines, as well as decreased tumor growth in a lung cancer mouse model. These results highlight a general function for HDLBP in the translation of ER-localized mRNAs and its relevance for tumor progression.

Peripheral nerves contain sensory and motor neuron axons coated by glial cells whose interplay ensures function, but molecular details are lacking. SNARE-proteins mediate the exchange and secretion of cargo by fusing vesicles with target organelles, but how glial SNAREs contribute to peripheral nerve function is largely unknown. We, here, identify non-neuronal Synaptobrevin (Syb) as the essential vesicular SNARE in Drosophila peripheral glia to insulate and metabolically supply neurons. We show that tetanus neurotoxin light chain (TeNT-LC), which potently inhibits SNARE-mediated exocytosis from neurons, also impairs peripheral nerve function when selectively expressed in glia, causing nerve disintegration, defective axonal transport, tetanic muscle hyperactivity, impaired locomotion, and lethality. While TeNT-LC disrupts neural function by cleaving neuronal Synaptobrevin (nSyb), it targets non-neuronal Synaptobrevin (Syb) in glia, which it cleaves at low rates: Glial knockdown of Syb (but not nSyb) phenocopied glial TeNT-LC expression whose effects were reverted by a TeNT-LC-insensitive Syb mutant. We link Syb-necessity to two distinct glial subtypes: Impairing Syb function in subperineurial glia disrupted nerve morphology, axonal transport, and locomotion, likely, because nerve-isolating septate junctions (SJs) could not form as essential SJ components (like the cell adhesion protein Neurexin-IV) were mistargeted. Interference with Syb in axon-encircling wrapping glia left nerve morphology and locomotion intact but impaired axonal transport, likely because neural metabolic supply was disrupted due to the mistargeting of metabolite shuffling monocarboxylate transporters. Our study identifies crucial roles of Syb in various glial subtypes to ensure glial-glial and glial-neural interplay needed for proper nerve function, animal motility, and survival.

Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with therapeutic potential applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of high-affinity nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent.

The hexameric ring structure of the type II AAA+ ATPases is considered as stable and permanent. Recently, the UBX domain-containing cofactors Arabidopsis thaliana PUX1 and human alveolar soft part sarcoma locus (ASPL) were reported to bind and disassemble the cognate AAA+ ATPases AtCDC48 and human p97. Here, we present two crystal structures related to these complexes: a truncated AtCDC48 (AtCDC48-ND1) and a hybrid complex containing human p97-ND1 and the UBX domain of plant PUX1 (p97-ND1:PUX1-UBX). These structures reveal close similarity between the human and plant AAA+ ATPases, but also highlight differences between disassembling and non-disassembling AAA+ ATPase cofactors. Based on an AtCDC48 disassembly assay with PUX1 and known crystal structures of the p97-bound human cofactor ASPL, we propose a general ATPase disassembly model. Thus, our structural and biophysical investigations provide detailed insight into the mechanism of AAA+ ATPase disassembly by UBX domain cofactors and suggest a general mode of regulating the cellular activity of these molecular machines.

Ribosome biogenesis is a fundamental multi-step cellular process that culminates in the formation of ribosomal subunits, whose production and modification are regulated by numerous biogenesis factors. In this study, we analyze physiologic prokaryotic ribosome biogenesis by isolating bona fide pre-50S subunits from an Escherichia coli strain with the biogenesis factor ObgE, affinity tagged at its native gene locus. Our integrative structural approach reveals a network of interacting biogenesis factors consisting of YjgA, RluD, RsfS, and ObgE on the immature pre-50S subunit. In addition, our study provides mechanistic insight into how the GTPase ObgE, in concert with other biogenesis factors, facilitates the maturation of the 50S functional core and reveals both conserved and divergent evolutionary features of ribosome biogenesis between prokaryotes and eukaryotes.

SummaryThe SAMHD1 triphosphohydrolase inhibits HIV-1 infection of myeloid and resting T cells by depleting dNTPs. To overcome SAMHD1, HIV-2 and some SIVs encode either of two lineages of the accessory protein Vpx that bind the SAMHD1 N or C terminus and redirect the host cullin-4 ubiquitin ligase to target SAMHD1 for proteasomal degradation. We present the ternary complex of Vpx from SIV that infects mandrills (SIVmnd-2) with the cullin-4 substrate receptor, DCAF1, and N-terminal and SAM domains from mandrill SAMHD1. The structure reveals details of Vpx lineage-specific targeting of SAMHD1 N-terminal “degron” sequences. Comparison with Vpx from SIV that infects sooty mangabeys (SIVsmm) complexed with SAMHD1-DCAF1 identifies molecular determinants directing Vpx lineages to N- or C-terminal SAMHD1 sequences. Inspection of the Vpx-DCAF1 interface also reveals conservation of Vpx with the evolutionally related HIV-1/SIV accessory protein Vpr. These data suggest a unified model for how Vpx and Vpr exploit DCAF1 to promote viral replication.

Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4(+) T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

Dynamin 1-like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.

SummaryGTPases of immunity-associated proteins (GIMAPs) are regulators of lymphocyte survival and homeostasis. We previously determined the structural basis of GTP-dependent GIMAP2 scaffold formation on lipid droplets. To understand how its GTP hydrolysis is activated, we screened for other GIMAPs on lipid droplets and identified GIMAP7. In contrast to GIMAP2, GIMAP7 displayed dimerization-stimulated GTP hydrolysis. The crystal structure of GTP-bound GIMAP7 showed a homodimer that assembled via the G domains, with the helical extensions protruding in opposite directions. We identified a catalytic arginine that is supplied to the opposing monomer to stimulate GTP hydrolysis. GIMAP7 also stimulated GTP hydrolysis by GIMAP2 via an analogous mechanism. Finally, we found GIMAP2 and GIMAP7 expression differentially regulated in several human T cell lymphoma lines. Our findings suggest that GTPase activity in the GIMAP family is controlled by homo- and heterodimerization. This may have implications for the differential roles of some GIMAPs in lymphocyte survival.

Stomatin proteins oligomerize at membranes and have been implicated in ion channel regulation and membrane trafficking. To obtain mechanistic insights into their function, we determined three crystal structures of the conserved stomatin domain of mouse stomatin that assembles into a banana-shaped dimer. We show that dimerization is crucial for the repression of acid-sensing ion channel 3 (ASIC3) activity. A hydrophobic pocket at the inside of the concave surface is open in the presence of an internal peptide ligand and closes in the absence of this ligand, and we demonstrate a function of this pocket in the inhibition of ASIC3 activity. In one crystal form, stomatin assembles via two conserved surfaces into a cylindrical oligomer, and these oligomerization surfaces are also essential for the inhibition of ASIC3-mediated currents. The assembly mode of stomatin uncovered in this study might serve as a model to understand oligomerization processes of related membrane-remodelling proteins, such as flotillin and prohibitin.

GTP ases of Immunity-Associated Proteins (GIMAPs) are a family of guanine nucleotide binding (G) proteins which are implicated in the regulation of apoptosis in lymphocytes. GIMAPs are composed of an amino-terminal G domain and carboxy-terminal extensions of varying size. Our recent biochemical and structural analysis of a representative GIMAP family member, GIMAP2, revealed the molecular basis of GTP-dependent oligomerization which involves two interfaces in the G domain. Whereas the amphipathic helix α7 in the C-terminal extension closely folds against the G domain in the GDP-bound state, it might be released in the GTP-bound state to assemble interaction partners. We also showed that the GIMAP2 oligomer functions at the surface of lipid droplets in a Jurkat T cell line. Here, we review our recent work and discuss the GIMAP2 oligomer as a GTP-dependent protein scaffold at the surface of lipid droplets controlling apoptosis.

GTPases of immunity-associated proteins (GIMAPs) are important regulators of T-cell death and survival. Here, the crystallization and data collection of three GIMAP2 constructs in various nucleotide-loaded states is described. Selenomethionine-substituted carboxy-terminally truncated GIMAP2 (amino-acid residues 1-260; GIMAP2(1-260)) in the nucleotide-free form crystallized in space group P2(1)2(1)2(1) and the crystals diffracted X-rays to 1.5 A resolution. The phase problem was solved using the single anomalous dispersion (SAD) protocol. GDP-bound GIMAP2(21-260) and GDP-bound GIMAP2(1-234) crystallized in space group P2(1)2(1)2(1) and the crystals diffracted X-rays to 2.9 and 1.7 A resolution, respectively. GTP-bound GIMAP2(1-234) crystallized in space group C222(1) and the crystals diffracted to 1.9 A resolution. These results will allow a detailed structural analysis of GIMAP2, which will provide insight into the architecture and function of the GIMAP family.

Mutations in SAMHD1 cause Aicardi–Goutières syndrome (AGS), a Mendelian inflammatory disease which displays remarkable clinical and biochemical overlap with congenital viral infection. SAMHD1 (SAM domain and HD domain-containing protein 1) has also been defined as an HIV-1 restriction-factor that, through a novel triphosphohydrolase activity, inhibits early stage HIV-1 replication in myeloid-derived dendritic cells (MDDCs), macrophages and resting CD4+ T-cells. The potent activity of SAMHD1 is likely to be the subject of a variety of regulatory mechanisms. Knowledge of proteins that interact with SAMHD1 may not only enhance our understanding of the pathogenesis of AGS, but may also provide further details on the link between the regulation of cellular dNTPs and HIV-1 restriction. In the present study, we used a yeast two-hybrid screen and pull-down analysis followed by MS to identify the eukaryotic elongation factor 1A1 (eEF1A1) as a potential interaction partner of SAMHD1. This interaction was confirmed by unbiased co-immunoprecipitation and demonstrated in situ by a proximity ligation assay (PLA). We show that this interaction is enhanced in mutant SAMHD1 cell lines and suggest that eEF1A1 may mediate SAMHD1 turnover by targeting it to the proteosome for degradation through association with Cullin4A and Rbx1.

In this study, we harness Whole Exome Sequencing (WES) to interrogate the underlying genetic cause in a patient cohort suffering from a Mendelian disease of immune dysregulation characterized by severe lymphopenia, splenomegaly, anemia, thrombocytopenia and liver failure. Bioinformatic analysis revealed novel mutations in a small GTPase that led to a near complete loss of mature protein in patient cells. Animal models lacking these genes develop a disease remarkably similar to that observed in the human patients, however, the molecular role of this GTPase in the immune system remains a fundamental gap in our knowledge. Due to a remarkable similarity in phenotypes between mice deficient for this small GTPase and T cell specific deletions of regulators of mTORC1, we hypothesized that this gene may be involved in the mTORC1 signaling pathway. We observe a robust association between this GTPase and members of the ragulator complex which is required for recruitment of mTORC1 to the lysosome and subsequent signalling. Furthermore, in human T cells deficient for this GTPase we observe increased phosphorylation of targets downstream of mTORC1. Overall, our data suggest that this GTPase is a novel T cell specific negative regulator of mTORC1 signalling. This research was supported by the Intramural Research Program of NIAID, NIH.

GTPases of immunity-associated proteins (GIMAPs) are a distinctive family of GTPases, which control apoptosis in lymphocytes and play a central role in lymphocyte maturation and lymphocyte-associated diseases. To explore their function and mechanism, we determined crystal structures of a representative member, GIMAP2, in different nucleotide-loading and oligomerization states. Nucleotide-free and GDP-bound GIMAP2 were monomeric and revealed a guanine nucleotide-binding domain of the TRAFAC (translation factor associated) class with a unique amphipathic helix α7 packing against switch II. In the absence of α7 and the presence of GTP, GIMAP2 oligomerized via two distinct interfaces in the crystal. GTP-induced stabilization of switch I mediates dimerization across the nucleotide-binding site, which also involves the GIMAP specificity motif and the nucleotide base. Structural rearrangements in switch II appear to induce the release of α7 allowing oligomerization to proceed via a second interface. The unique architecture of the linear oligomer was confirmed by mutagenesis. Furthermore, we showed a function for the GIMAP2 oligomer at the surface of lipid droplets. Although earlier studies indicated that GIMAPs are related to the septins, the current structure also revealed a strikingly similar nucleotide coordination and dimerization mode as in the dynamin GTPase. Based on this, we reexamined the relationships of the septin- and dynamin-like GTPases and demonstrate that these are likely to have emerged from a common membrane-associated dimerizing ancestor. This ancestral property appears to be critical for the role of GIMAPs as nucleotide-regulated scaffolds on intracellular membranes.

The inhibition of carbohydrate-protein interactions by tailored multivalent ligands is a powerful strategy for the treatment of many human diseases. Crucial for the success of this approach is an understanding of the molecular mechanisms as to how a binding enhancement of a multivalent ligand is achieved. We have synthesized a series of multivalent N-acetylglucosamine (GlcNAc) derivatives and studied their interaction with the plant lectin wheat germ agglutinin (WGA) by an enzyme-linked lectin assay (ELLA) and X-ray crystallography. The solution conformation of one ligand was determined by NMR spectroscopy. Employing a GlcNAc carbamate motif with alpha-configuration and by systematic variation of the spacer length, we were able to identify divalent ligands with unprecedented high WGA binding potency. The best divalent ligand has an IC(50) value of 9.8 microM (ELLA) corresponding to a relative potency of 2350 (1170 on a valency-corrected basis, i.e., per mol sugar contained) compared to free GlcNAc. X-ray crystallography of the complex of WGA and the second best, closely related divalent ligand explains this activity. Four divalent molecules simultaneously bind to WGA with each ligand bridging adjacent binding sites. This shows for the first time that all eight sugar binding sites of the WGA dimer are simultaneously functional. We also report a tetravalent neoglycopeptide with an IC(50) value of 0.9 microM being 25,500 times higher than that of GlcNAc (6400 times per contained sugar) and the X-ray structure analysis of its complex with glutaraldehyde-cross-linked WGA. Comparison of the crystal structure and the solution NMR structure of the neoglycopeptide as well as results from the ELLA suggest that the conformation of the glycopeptide in solution is already preorganized in a way supporting multivalent binding to the protein. Our findings show that bridging adjacent protein binding sites by multivalent ligands is a valid strategy to find high-affinity protein ligands and that even subtle changes of the linker structure can have a significant impact on the binding affinity.

SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form “long-lived” enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells., Author Summary SAMHD1 is a restriction factor that blocks infection of certain immune cells by HIV-1. It was discovered to be an enzyme that catalyses the breakdown of dNTPs, suggesting that it inhibits HIV-1 replication by reducing cellular dNTP pools to such low levels that reverse transcriptase cannot function. However, recently, alternative mechanisms have been proposed. SAMHD1 is also regulated by phosphorylation, although the effects of phosphorylation on protein function are unclear. In order to address these issues, we carried out combined structural and virological studies and have demonstrated that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form “long-lived” enzymatically competent tetramers. Disrupting the tetramer in various ways always abolished restriction but had differing effects on enzyme activity in vitro. SAMHD1 phosphorylation also prevented restriction and tetramer formation but without affecting enzyme catalysis under steady-state dNTP conditions. However phosphorylated SAMHD1 was unable to catalyse dNTP turnover at very low nucleotide levels that more accurately represent conditions in the cells in which restriction takes place. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity and substantiate that degradation of dNTPs by SAMHD1 is sufficient to restrict HIV-1.