A central goal of HIV-1 vaccine research is the elicitation of antibodies capable of neutralizing diverse primary isolates of HIV-1. Here we show that focusing the immune response to exposed N-terminal residues of the fusion peptide, a critical component of the viral entry machinery and the epitope of antibodies elicited by HIV-1 infection, through immunization with fusion peptide-coupled carriers and prefusion stabilized envelope trimers, induces cross-clade neutralizing responses. In mice, these immunogens elicited monoclonal antibodies capable of neutralizing up to 31% of a cross-clade panel of 208 HIV-1 strains. Crystal and cryoelectron microscopy structures of these antibodies revealed fusion peptide conformational diversity as a molecular explanation for the cross-clade neutralization. Immunization of guinea pigs and rhesus macaques induced similarly broad fusion peptide-directed neutralizing responses, suggesting translatability. The N terminus of the HIV-1 fusion peptide is thus a promising target of vaccine efforts aimed at eliciting broadly neutralizing antibodies.

Summary The insulin receptor (IR) is a dimeric protein that plays a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels1,2. IR dysfunctions have been associated with many diseases, including diabetes, cancer, and Alzheimer’s1,3,4. The primary sequence has been known since the 1980s5, and is composed of an extracellular portion (ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Insulin binding to the dimeric ECD triggers kinase domain auto-phosphorylation and subsequent activation of downstream signaling molecules. Biochemical and mutagenesis data have identified two putative insulin binding sites (S1 and S2)6. While insulin bound to an ECD fragment containing S1 and the apo ectodomain have been characterized structurally7,8, details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single particle cryoEM reconstructions for the 1:2 (4.3 Å) and 1:1 (7.4 Å) IR ECD dimer:Insulin complexes. The symmetric 4.3 Å structure shows two insulin molecules per dimer, each bound between the Leucine-rich sub domain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the S1 binding interactions and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative sub domain orientations and triggers downstream signal propagation.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

Most protein particles prepared in vitreous ice for single particle cryo-electron microscopy are adsorbed to air-water or substrate-water interfaces, potentially causing particles to adopt preferred orientations. Using the Spotiton robot and nanowire grids, we can reduce some of the deleterious effects of the air-water interface by decreasing the dwell time of particles in thin liquid films. We demonstrate this by using single particle cryoEM and cryoET on three biological samples.

Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

SUMMARY The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Classically, G protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (βarr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-βarr 'megaplex'. Nevertheless, the megaplex's molecular architecture remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine βarr to the core and phosphorylated tail, respectively, of a single active human chimeric β 2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (β 2 V 2 R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and βarr. Our findings provide a structural basis for GPCR-mediated sustained, internalized G protein signaling. Nguyen et al.

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structurebased mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.inflammation | protein structure | protein filament | ASC speck | innate immune response