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There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technology has a competitive advantage over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo –matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallography in a time span of 6–12 months.

Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity. Recent studies revealed that G protein-coupled receptors rapidly interconvert between multiple states. Here, authors use the kappa opioid receptor (KOR) and show how two state-dependent nanobodies provide real-time reporting of ligand stabilized states with KOR and other GPCRs.

Type A γ-aminobutyric acid (GABA A ) receptors are pentameric ligand-gated ion channels and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system 1 , 2 . Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia 3 , 4 . Among the numerous assemblies that are theoretically possible, the most prevalent in the brain are the α1β2/3γ2 GABA A receptors 5 . The β3 subunit has an important role in maintaining inhibitory tone, and the expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in β1–β3 triple knockout neurons 6 . So far, efforts to generate accurate structural models for heteromeric GABA A receptors have been hampered by the use of engineered receptors and the presence of detergents 7 – 9 . Notably, some recent cryo-electron microscopy reconstructions have reported ‘collapsed’ conformations 8 , 9 ; however, these disagree with the structure of the prototypical pentameric ligand-gated ion channel the Torpedo nicotinic acetylcholine receptor 10 , 11 , the large body of structural work on homologous homopentameric receptor variants 12 and the logic of an ion-channel architecture. Here we present a high-resolution cryo-electron microscopy structure of the full-length human α1β3γ2L—a major synaptic GABA A receptor isoform—that is functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator ‘megabody’ and is in a desensitized conformation. Each GABA A receptor pentamer contains two phosphatidylinositol-4,5-bisphosphate molecules, the head groups of which occupy positively charged pockets in the intracellular juxtamembrane regions of α1 subunits. Beyond this level, the intracellular M3–M4 loops are largely disordered, possibly because interacting post-synaptic proteins are not present. This structure illustrates the molecular principles of heteromeric GABA A receptor organization and provides a reference framework for future mechanistic investigations of GABAergic signalling and pharmacology. A high-resolution cryo-electron microscopy structure is reported for the full-length human α1β3γ2L GABA A receptor, functionally reconstituted in lipid nanodiscs.

Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water–air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.Megabodies, built by grafting nanobodies onto larger protein scaffolds, help alleviate problems of particle size and preferential orientation at the water–air interfaces during cryo-EM based structure determination experiments and are shown to be generalizable to soluble and membrane-bound proteins.

Hyaluronan is an acidic heteropolysaccharide comprising alternating N -acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix 1 . The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis 2 . Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors 3 , 4 . Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body. A cryo-electron microscopy analysis reveals how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore.

Crystal structures and functional assays of a chimeric GABA A receptor in apo and pregnanolone-bound states reveal how neurosteroid binding alters receptor conformation to modulate channel opening. Type A γ-aminobutyric acid receptors (GABA A Rs) are the principal mediators of inhibitory neurotransmission in the human brain. Endogenous neurosteroids interact with GABA A Rs to regulate acute and chronic anxiety and are potent sedative, analgesic, anticonvulsant and anesthetic agents. Their mode of binding and mechanism of receptor potentiation, however, remain unknown. Here we report crystal structures of a chimeric GABA A R construct in apo and pregnanolone-bound states. The neurosteroid-binding site is mechanically coupled to the helices lining the ion channel pore and modulates the desensitization-gate conformation. We demonstrate that the equivalent site is responsible for physiological, heteromeric GABA A R potentiation and explain the contrasting modulatory properties of 3a versus 3b neurosteroid epimers. These results illustrate how peripheral lipid ligands can regulate the desensitization gate of GABA A Rs, a process of broad relevance to pentameric ligand-gated ion channels.

The integrity of a cell’s proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC. Tagging of the endogenous type II chaperonin TRiC complex using CRISPR knock-in enables its purification for cryo-EM. A series of structures reveal the fate of substrates and co-chaperones inside the TRiC chamber to uncover its inner workings.

Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development. Influenza viruses carry their own RNAdependent RNA-polymerase that is highly conserved and a promising anti-viral target. Combining functional and structural data, Keown et al. characterise the inhibitory effect of nanobodies on 1918 pandemic H1N1 influenza strain polymerase complex and identify sensitive sites interfering with polymerase activity in vitro.

The Gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a deadly disease mostly affecting wildlife and livestock, as well as representing a bioterrorism threat. Its cell surface is covered by the mutually exclusive S-layers Sap and EA1, found in early and late growth phases, respectively. Here we report the nanobody-based structural characterization of EA1 and its native lattice contacts. The EA1 assembly domain consists of 6 immunoglobulin-like domains, where three calcium-binding sites structure interdomain contacts that allow monomers to adopt their assembly-competent conformation. Nanobody-induced depolymerization of EA1 S-layers results in surface defects, membrane blebbing and cell lysis under hypotonic conditions, indicating that S-layers provide additional mechanical stability to the cell wall. Taken together, we report a complete model of the EA1 S-layer and present a set of nanobodies that may have therapeutic potential against Bacillus anthracis . S-layers form continuous protein lattices on the surface of bacteria. Here, authors use S-layer depolymerizing nanobodies to solve the structure of the EA1 S-layer in the pathogen Bacillus anthracis and show its role as cell wall supportive structure”

L-amino acid transporters (LATs) play key roles in human physiology and are implicated in several human pathologies. LATs are asymmetric amino acid exchangers where the low apparent affinity cytoplasmic side controls the exchange of substrates with high apparent affinity on the extracellular side. Here, we report the crystal structures of an LAT, the bacterial alanine-serine-cysteine exchanger (BasC), in a non-occluded inward-facing conformation in both apo and substrate-bound states. We crystallized BasC in complex with a nanobody, which blocks the transporter from the intracellular side, thus unveiling the sidedness of the substrate interaction of BasC. Two conserved residues in human LATs, Tyr 236 and Lys 154, are located in equivalent positions to the Na1 and Na2 sites of sodium-dependent APC superfamily transporters. Functional studies and molecular dynamics (MD) calculations reveal that these residues are key for the asymmetric substrate interaction of BasC and in the homologous human transporter Asc-1. L-Amino acid Transporters (LATs) are asymmetric amino acid exchangers. Here the authors determine the crystal structure of a prokaryotic LAT, the alanine-serine-cysteine exchanger (BasC) and identify key residues for asymmetric substrate interaction in both BasC and the homologous human transporter Asc-1 through functional studies.