Structural basis of second-generation HIV integrase inhibitor action and viral resistance

Despite worldwide prescription, the mechanistic basis for superiority of second-generation HIV integrase (IN) strand transfer inhibitors (INSTIs) is poorly understood. We use single-particle cryo-electron microscopy to visualize the mode of action of the advanced INSTIs dolutegravir and bictegravir at near atomic resolution. Q148H/G140S amino acid substitutions in IN that pervade clinical INSTI failure perturb optimal magnesium ion coordination in the enzyme active site. The expanded chemical scaffolds of second-generation compounds mediate interactions with the protein backbone, which are critical for antagonizing Q148H/G140S mutant virus. Our results reveal that binding to magnesium ions underpins a fundamental weakness of the INSTI pharmacophore that is exploited by the virus to engender resistance and provide a structural framework for the development of this important class of anti-HIV/AIDS therapeutics

Cryo-EM structures of the XPF-ERCC1 endonuclease reveal how DNA-junction engagement disrupts an auto-inhibited conformation

The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). How XPF-ERCC1 is catalytically activated by DNA junction substrates is not currently understood. Here we report cryo-electron microscopy structures of both DNA-free and DNA-bound human XPF-ERCC1. DNA-free XPF-ERCC1 adopts an auto-inhibited conformation in which the XPF helical domain masks the ERCC1 (HhH)2 domain and restricts access to the XPF catalytic site. DNA junction engagement releases the ERCC1 (HhH)2 domain to couple with the XPF-ERCC1 nuclease/nuclease-like domains. Structure-function data indicate xeroderma pigmentosum patient mutations frequently compromise the structural integrity of XPF-ERCC1. Fanconi anaemia patient mutations in XPF often display substantial in-vitro activity but are resistant to activation by ICLR recruitment factor SLX4. Our data provide insights into XPF-ERCC1 architecture and catalytic activation

Structural basis for Fullerene geometry in a human endogenous retrovirus capsid

The HML2 (HERV-K) group constitutes the most recently acquired family of human endogenous retroviruses, with many proviruses less than one million years old. Many maintain intact open reading frames and provirus expression together with HML2 particle formation are observed in early stage human embryo development and are associated with pluripotency as well as inflammatory disease, cancers and HIV-1 infection. Here, we reconstruct the core structural protein (CA) of an HML2 retrovirus, assemble particles in vitro and employ single particle cryogenic electron microscopy (cryo-EM) to determine structures of four classes of CA Fullerene shell assemblies. These icosahedral and capsular assemblies reveal at high-resolution the molecular interactions that allow CA to form both pentamers and hexamers and show how invariant pentamers and structurally plastic hexamers associate to form the unique polyhedral structures found in retroviral cores

Platform-directed allostery and quaternary structure dynamics of SAMHD1 catalysis

SAMHD1 regulates cellular nucleotide homeostasis, controlling dNTP levels by catalysing their hydrolysis into 2’-deoxynucleosides and triphosphate. In differentiated CD4+ macrophage and resting T-cells SAMHD1 activity results in the inhibition of HIV-1 infection through a dNTP blockade. In cancer, SAMHD1 desensitizes cells to nucleoside-analogue chemotherapies. Here we employ time-resolved cryogenic-EM imaging and single-particle analysis to visualise assembly, allostery and catalysis by this multi-subunit enzyme. Our observations reveal how dynamic conformational changes in the SAMHD1 quaternary structure drive the catalytic cycle. We capture five states at high-resolution in a live catalytic reaction, revealing how allosteric activators support assembly of a stable SAMHD1 tetrameric core and how catalysis is driven by the opening and closing of active sites through pairwise coupling of active sites and order-disorder transitions in regulatory domains. This direct visualisation of enzyme catalysis dynamics within an allostery-stabilised platform sets a precedent for mechanistic studies into the regulation of multi-subunit enzymes

The CAMELS-CL dataset: catchment attributes and meteorology for large sample studies – Chile dataset

We introduce the first catchment dataset for large sample studies in Chile. This dataset includes 516 catchments; it covers particularly wide latitude (17.8 to 55.0∘ S) and elevation (0 to 6993 m a.s.l.) ranges, and it relies on multiple data sources (including ground data, remote-sensed products and reanalyses) to characterise the hydroclimatic conditions and landscape of a region where in situ measurements are scarce. For each catchment, the dataset provides boundaries, daily streamflow records and basin-averaged daily time series of precipitation (from one national and three global datasets), maximum, minimum and mean temperatures, potential evapotranspiration (PET; from two datasets), and snow water equivalent. We calculated hydro-climatological indices using these time series, and leveraged diverse data sources to extract topographic, geological and land cover features. Relying on publicly available reservoirs and water rights data for the country, we estimated the degree of anthropic intervention within the catchments. To facilitate the use of this dataset and promote common standards in large sample studies, we computed most catchment attributes introduced by Addor et al. (2017) in their Catchment Attributes and MEteorology for Large-sample Studies (CAMELS) dataset, and added several others. We used the dataset presented here (named CAMELS-CL) to characterise regional variations in hydroclimatic conditions over Chile and to explore how basin behaviour is influenced by catchment attributes and water extractions. Further, CAMELS-CL enabled us to analyse biases and uncertainties in basin-wide precipitation and PET. The characterisation of catchment water balances revealed large discrepancies between precipitation products in arid regions and a systematic precipitation underestimation in headwater mountain catchments (high elevations and steep slopes) over humid regions. We evaluated PET products based on ground data and found a fairly good performance of both products in humid regions (r>0.91) and lower correlation (r<0.76) in hyper-arid regions. Further, the satellite-based PET showed a consistent overestimation of observation-based PET. Finally, we explored local anomalies in catchment response by analysing the relationship between hydrological signatures and an attribute characterising the level of anthropic interventions. We showed that larger anthropic interventions are correlated with lower than normal annual flows, runoff ratios, elasticity of runoff with respect to precipitation, and flashiness of runoff, especially in arid catchments. CAMELS-CL provides unprecedented information on catchments in a region largely underrepresented in large sample studies. This effort is part of an international initiative to create multi-national large sample datasets freely available for the community. CAMELS-CL can be visualised from http://camels.cr2.cl and downloaded from https://doi.pangaea.de/10.1594/PANGAEA.894885

Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer

Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase gains access to the scissile phosphodiester bonds by lifting DNA off the histone octamer at the site of integration. To clarify the mechanism of DNA looping by integrase, we determined a 3.9 Å resolution structure of the prototype foamy virus intasome engaged with a nucleosome core particle. The structural data along with complementary single-molecule Förster resonance energy transfer measurements reveal twisting and sliding of the nucleosomal DNA arm proximal to the integration site. Sliding the nucleosomal DNA by approximately two base pairs along the histone octamer accommodates the necessary DNA lifting from the histone H2A-H2B subunits to allow engagement with the intasome. Thus, retroviral integration into nucleosomes involves the looping-and-sliding mechanism for nucleosomal DNA repositioning, bearing unexpected similarities to chromatin remodelers

Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome

Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Intersubunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitr

A bipartite structural organization defines the SERINC family of HIV-1 restriction factors

The human integral membrane protein SERINC5 potently restricts HIV-1 infectivity and sensitizes the virus to antibody-mediated neutralization. Here, using cryo-EM, we determine the structures of human SERINC5 and its orthologue from Drosophila melanogaster at subnanometer and near-atomic resolution, respectively. The structures reveal a novel fold comprised of ten transmembrane helices organized into two subdomains and bisected by a long diagonal helix. A lipid binding groove and clusters of conserved residues highlight potential functional sites. A structure-based mutagenesis scan identified surface-exposed regions and the interface between the subdomains of SERINC5 as critical for HIV-1-restriction activity. The same regions are also important for viral sensitization to neutralizing antibodies, directly linking the antiviral activity of SERINC5 with remodeling of the HIV-1 envelope glycoprotein

A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands

SARS-CoV-2 can recruit a haem metabolite to evade antibody immunity.

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through the recruitment of a metabolite