Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms.

Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cyro-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.

Anti-V1/V3-glycan broadly HIV-1 neutralizing antibodies in a post-treatment controller.

HIV-1 broadly neutralizing antibodies (bNAbs) can decrease viremia but are usually unable to counteract autologous viruses escaping the antibody pressure. Nonetheless, bNAbs may contribute to natural HIV-1 control in individuals off antiretroviral therapy (ART). Here, we describe a bNAb B cell lineage elicited in a post-treatment controller (PTC) that exhibits broad seroneutralization and show that a representative antibody from this lineage, EPTC112, targets a quaternary epitope in the glycan-V3 loop supersite of the HIV-1 envelope glycoprotein. The cryo-EM structure of EPTC112 complexed with soluble BG505 SOSIP.664 envelope trimers revealed interactions with N301- and N156-branched N-glycans and the 324GDIR327 V3 loop motif. Although the sole contemporaneous virus circulating in this PTC was resistant to EPTC112, it was potently neutralized by autologous plasma IgG antibodies. Our findings illuminate how cross-neutralizing antibodies can alter the HIV-1 infection course in PTCs and may control viremia off-ART, supporting their role in functional HIV-1 cure strategies.

Cryo-EM and Molecular Dynamics Simulations Reveal Hidden Conformational Dynamics Controlling Ammonia Transport in Human Asparagine Synthetase.

How dynamical motions in enzymes might be linked to catalytic function is of significant general interest, although almost all relevant experimental data, to date, has been obtained for enzymes with a single active site. Recent advances in X-ray crystallography and cryogenic electron microscopy offer the promise of elucidating dynamical motions for proteins that are not amenable to study using solution-phase NMR methods. Here we use 3D variability analysis (3DVA) of an EM structure for human asparagine synthetase (ASNS) in combination with atomistic molecular dynamics (MD) simulations to detail how dynamic motions of a single side chain mediates interconversion of the open and closed forms of a catalytically relevant intramolecular tunnel, thereby regulating catalytic function. Our 3DVA results are consistent with those obtained independently from MD simulations, which further suggest that formation of a key reaction intermediate acts to stabilize the open form of the tunnel in ASNS to permit ammonia translocation and asparagine formation. This conformational selection mechanism for regulating ammonia transfer in human ASNS contrasts sharply with those employed in other glutamine-dependent amidotransferases that possess a homologous glutaminase domain. Our work illustrates the power of cryo-EM to identify localized conformational changes and hence dissect the conformational landscape of large proteins. When combined with MD simulations, 3DVA is a powerful approach to understanding how conformational dynamics regulate function in metabolic enzymes with multiple active sites.

Tunneling nanotubes provide a route for SARS-CoV-2 spreading.

Neurological manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection represent a major issue in long coronavirus disease. How SARS-CoV-2 gains access to the brain and how infection leads to neurological symptoms are not clear because the principal means of viral entry by endocytosis, the angiotensin-converting enzyme 2 receptor, are barely detectable in the brain. We report that human neuronal cells, nonpermissive to infection through the endocytic pathway, can be infected when cocultured with permissive infected epithelial cells. SARS-CoV-2 induces the formation of tunneling nanotubes (TNTs) and exploits this route to spread to uninfected cells. In cellulo correlative fluorescence and cryo-electron tomography reveal that SARS-CoV-2 is associated with TNTs between permissive cells. Furthermore, multiple vesicular structures such as double-membrane vesicles, sites of viral replication, are observed inside TNTs between permissive and nonpermissive cells. Our data highlight a previously unknown mechanism of SARS-CoV-2 spreading, likely used as a route to invade nonpermissive cells and potentiate infection in permissive cells.

Spindle-shaped archaeal viruses evolved from rod-shaped ancestors to package a larger genome.

Spindle- or lemon-shaped viruses infect archaea in diverse environments. Due to the highly pleomorphic nature of these virions, which can be found with cylindrical tails emanating from the spindle-shaped body, structural studies of these capsids have been challenging. We have determined the atomic structure of the capsid of Sulfolobus monocaudavirus 1, a virus that infects hosts living in nearly boiling acid. A highly hydrophobic protein, likely integrated into the host membrane before the virions assemble, forms 7 strands that slide past each other in both the tails and the spindle body. We observe the discrete steps that occur as the tail tubes expand, and these are due to highly conserved quasiequivalent interactions with neighboring subunits maintained despite significant diameter changes. Our results show how helical assemblies can vary their diameters, becoming nearly spherical to package a larger genome and suggest how all spindle-shaped viruses have evolved from archaeal rod-like viruses.

Atomic structure of Lanreotide nanotubes revealed by cryo-EM.

Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different from the molecular model, largely based on X-ray fiber diffraction, proposed 20 y ago. Comparison of the nanotube with a crystal structure at 0.83-Å resolution of a Lanreotide derivative highlights the polymorphism for this peptide family. This work shows once again that higher-order assemblies formed by even well-characterized small peptides are very difficult to predict.

Cryptococcus extracellular vesicles properties and their use as vaccine platforms.

Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii. Using cutting-edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein-based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analysing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane-bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection.

The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy.

The structure of the infectious prion protein (PrPSc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc and its N-terminally truncated variant, PrP 27-30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27-30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 Å3, predicted the height of each PrP 27-30 molecule as ~17.7 Å. Together, the data indicate a four-rung ß-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.

Archaeal flagellin combines a bacterial type IV pilin domain with an Ig-like domain.

The bacterial flagellar apparatus, which involves ∼40 different proteins, has been a model system for understanding motility and chemotaxis. The bacterial flagellar filament, largely composed of a single protein, flagellin, has been a model for understanding protein assembly. This system has no homology to the eukaryotic flagellum, in which the filament alone, composed of a microtubule-based axoneme, contains more than 400 different proteins. The archaeal flagellar system is simpler still, in some cases having ∼13 different proteins with a single flagellar filament protein. The archaeal flagellar system has no homology to the bacterial one and must have arisen by convergent evolution. However, it has been understood that the N-terminal domain of the archaeal flagellin is a homolog of the N-terminal domain of bacterial type IV pilin, showing once again how proteins can be repurposed in evolution for different functions. Using cryo-EM, we have been able to generate a nearly complete atomic model for a flagellar-like filament of the archaeon Ignicoccus hospitalis from a reconstruction at ∼4-Å resolution. We can now show that the archaeal flagellar filament contains a ß-sandwich, previously seen in the FlaF protein that forms the anchor for the archaeal flagellar filament. In contrast to the bacterial flagellar filament, where the outer globular domains make no contact with each other and are not necessary for either assembly or motility, the archaeal flagellin outer domains make extensive contacts with each other that largely determine the interesting mechanical properties of these filaments, allowing these filaments to flex.

Extensive subunit contacts underpin herpesvirus capsid stability and interior-to-exterior allostery.

The herpesvirus capsid is a complex protein assembly that includes hundreds of copies of four major subunits and lesser numbers of several minor proteins, all of which are essential for infectivity. Cryo-electron microscopy is uniquely suited for studying interactions that govern the assembly and function of such large functional complexes. Here we report two high-quality capsid structures, from human herpes simplex virus type 1 (HSV-1) and the animal pseudorabies virus (PRV), imaged inside intact virions at ~7-Å resolution. From these, we developed a complete model of subunit and domain organization and identified extensive networks of subunit contacts that underpin capsid stability and form a pathway that may signal the completion of DNA packaging from the capsid interior to outer surface, thereby initiating nuclear egress. Differences in the folding and orientation of subunit domains between herpesvirus capsids suggest that common elements have been modified for specific functions.

FEI's direct electron detector developments: Embarking on a revolution in cryo-TEM.

In early 2011 FEI Company launched the "Falcon", its first commercial direct electron detector product intended for application in 3-D electron microscopy in the life sciences. In this paper we discuss the principle of direct electron detection and its implementation in Falcon cameras. We describe the signal formation in the sensor and its impact on the detection quantum efficiency (DQE) of the sensor. Insights into the signal formation led us to improved camera designs. Three significant improvements are discussed. (1) Back thinning of the sensor. This is implemented in the second-generation Falcon (Falcon 2), where the sensor thickness is reduced to 50 µm, and in the latest generation Falcon 3 detector with further back-thinning down to 30 µm. (2) The introduction of electron counting, a signal processing technology implemented in Falcon 3. (3) Dose fractionation mode, which allows the user to access intermediate results during the illumination of the sample.

Structural snapshots of actively translating human ribosomes.

Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.

Near-atomic resolution for one state of F-actin.

Actin functions as a helical polymer, F-actin, but attempts to build an atomic model for this filament have been hampered by the fact that the filament cannot be crystallized and by structural heterogeneity. We have used a direct electron detector, cryo-electron microscopy, and the forces imposed on actin filaments in thin films to reconstruct one state of the filament at 4.7 Å resolution, which allows for building a reliable pseudo-atomic model of F-actin. We also report a different state of the filament where actin protomers adopt a conformation observed in the crystal structure of the G-actin-profilin complex with an open ATP-binding cleft. Comparison of the two structural states provides insights into ATP-hydrolysis and filament dynamics. The atomic model provides a framework for understanding why every buried residue in actin has been under intense selective pressure.

Tough stimuli-responsive supramolecular hydrogels with hydrogen-bonding network junctions.

Hydrogels were prepared with physical cross-links comprising 2-ureido-4[1H]-pyrimidinone (UPy) hydrogen-bonding units within the backbone of segmented amphiphilic macromolecules having hydrophilic poly(ethylene glycol) (PEG). The bulk materials adopt nanoscopic physical cross-links composed of UPy-UPy dimers embedded in segregated hydrophobic domains dispersed within the PEG matrix as comfirmed by cryo-electron microscopy. The amphiphilic network was swollen with high weight fractions of water (w(H2O) ≈ 0.8) owing to the high PEG weight fraction within the pristine polymers (w(PEG) ≈ 0.9). Two different PEG chain lengths were investigated and illustrate the corresponding consequences of cross-link density on mechanical properties. The resulting hydrogels exhibited high strength and resilience upon deformation, consistent with a microphase separated network, in which the UPy-UPy interactions were adequately shielded within hydrophobic nanoscale pockets that maintain the network despite extensive water content. The cumulative result is a series of tough hydrogels with tunable mechanical properties and tractable synthetic preparation and processing. Furthermore, the melting transition of PEG in the dry polymer was shown to be an effective stimulus for shape memory behavior.

Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae.

The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression-maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes.

Physiological implications of NTBI uptake by T lymphocytes.

In iron overload disorders a significant fraction of the total iron circulates in the plasma as low molecular weight complexes not bound to transferrin, known as non-transferrin-bound iron (NTBI). By catalyzing the formation of free radicals, NTBI accumulation results in oxidative stress and cellular damage, being a major cause of organ toxicity. NTBI is rapidly and preferentially cleared from circulation by the liver and the myocardium, the main disease targets in iron overload conditions. We have recently demonstrated that human peripheral blood T lymphocytes take up NTBI in vitro, with a pattern that resembles that of hepatocytes. Since T lymphocytes constitute a numerically important component of the circulating cell pool, these findings support a putative role for this cell type in the systemic protection against iron toxicity. Here we tested the hypothesis that the circulating peripheral blood T lymphocyte pool constitutes an important storage compartment for NTBI and is thus a modifier of NTBI deposition in target organs. First we show that NTBI uptake by human T lymphocytes increases the expression of the iron-storage protein ferritin and of the iron exporter ferroportin via an IRE-dependent mechanism. NTBI retention by T lymphocytes is shown to be critically controlled by the hepcidin-mediated modulation of ferroportin both in vitro and in vivo. Finally, the protective effect of T lymphocytes was tested by analyzing the patterns of iron accumulation in the T lymphocyte-deficient mouse model Foxn1(nu) before and after reconstitution with T lymphocytes by adoptive transfer. The results confirmed a significant increase of liver and pancreas iron accumulation in T lymphocyte-deficient mice. NTBI accumulation in the liver and spleen was prevented by reconstitution with syngeneic T lymphocytes. Altogether, our results demonstrate that T lymphocytes are important components of a circulating "NTBI storage compartment" and show its physiological relevance as a modifier of tissue iron overload.

Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes.

Inflammasomes elicit host defense inside cells by activating caspase-1 for cytokine maturation and cell death. AIM2 and NLRP3 are representative sensor proteins in two major families of inflammasomes. The adaptor protein ASC bridges the sensor proteins and caspase-1 to form ternary inflammasome complexes, achieved through pyrin domain (PYD) interactions between sensors and ASC and through caspase activation and recruitment domain (CARD) interactions between ASC and caspase-1. We found that PYD and CARD both form filaments. Activated AIM2 and NLRP3 nucleate PYD filaments of ASC, which, in turn, cluster the CARD of ASC. ASC thus nucleates CARD filaments of caspase-1, leading to proximity-induced activation. Endogenous NLRP3 inflammasome is also filamentous. The cryoelectron microscopy structure of ASC(PYD) filament at near-atomic resolution provides a template for homo- and hetero-PYD/PYD associations, as confirmed by structure-guided mutagenesis. We propose that ASC-dependent inflammasomes in both families share a unified assembly mechanism that involves two successive steps of nucleation-induced polymerization. PAPERFLICK:

Non-transferrin-bound iron (NTBI) uptake by T lymphocytes: evidence for the selective acquisition of oligomeric ferric citrate species.

Iron is an essential nutrient in several biological processes such as oxygen transport, DNA replication and erythropoiesis. Plasma iron normally circulates bound to transferrin. In iron overload disorders, however, iron concentrations exceed transferrin binding capacity and iron appears complexed with low molecular weight molecules, known as non-transferrin-bound iron (NTBI). NTBI is responsible for the toxicity associated with iron-overload pathologies but the mechanisms leading to NTBI uptake are not fully understood. Here we show for the first time that T lymphocytes are able to take up and accumulate NTBI in a manner that resembles that of hepatocytes. Moreover, we show that both hepatocytes and T lymphocytes take up the oligomeric Fe3Cit3 preferentially to other iron-citrate species, suggesting the existence of a selective NTBI carrier. These results provide a tool for the identification of the still elusive ferric-citrate cellular carrier and may also open a new pathway towards the design of more efficient iron chelators for the treatment of iron overload disorders.

Perspectives on electron cryo-tomography of vitreous cryo-sections.

A major objective of modern structural biology is to appreciate the cellular organization by elucidating the spatial arrangement of macromolecular complexes within a cell. Cryogenic sample preparation, combined with cryo-ultramicrotomy, enables large cells and pieces of biological tissues to be thinned for electron cryo-tomography, which provides a three-dimensional view of the biological sample. There are, however, limitations associated with the technique that must be realized, addressed and overcome for the procedure to become mainstream. Here, we provide perspectives on the continued advancements in cryogenic sample preparation for vitreous cryo-sectioning, image collection and post-image processing that have expanded the attainable information limit within the three-dimensional reconstructions of cells and pieces of biological tissues.