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K2P channels are important regulators of cellular electrical activity. Here the authors show how nanobody fragments can be used to detect and modulate TREK2 K2P channel activity to provide insight into the mechanism of gating.

Mutations in glucocerebrosidase lead to Parkinson’s and Gaucher disease. This study reports the characterization of nanobodies that improve glucocerebrosidase functionality using an allosteric mechanism. This paves the way for alternative therapeutic routes.

Here, the structure of SLC19A3, an essential human transport protein for vitamin B₁, has been determined in different conformations and bound to its substrate and different drug molecules. The researchers provide deep molecular insights into the transport cycle and drug interactions of this critical protein.

Protein-protein interactions are central in cell metabolism but research tools for their characterization are missing. Here, the authors introduce strategies for the discovery of nanobodies that modulate the SOS1•RAS complex formation.

The Wnt receptor Frizzled (FZD) family is crucial for both canonical (β-catenin dependent) and non-canonical (β-catenin independent) Wnt signalling. Here, the authors present the structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nbs), elucidating extracellular and intracellular interaction surfaces of functional and potentially therapeutic significance.

Kuhm et al. reveal how human guanylate-binding protein 1 (GBP1) dimers self-associate to coat bacterial pathogens and uncover a guanosine triphosphate hydrolase-dependent membrane-remodeling activity of GBP1 that is crucial for intracellular immunity

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”

The structures of the retrovirus-derived human syncytin-1 and suppressyn in complexes with their shared receptor reveal an ancient cellular recognition mechanism that underlies key morphological and immunological functions in placenta.

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.

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.

Structural studies of human Na⁺–taurocholate co-transporting polypeptide in complex with nanobodies reveal mechanisms for bile salts transport and HBV recognition involving an open-pore intermediate state.

G protein-coupled receptors (GPCRs) are involved in many physiological processes and are targets of intense drug discovery research. Here, the authors describe llama-derived nanobodies that allosterically modulate rhodopsin, a prototypical GPCR.

The leading cause of cystic fibrosis is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). Here authors we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein.

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.

Cryo-electron microscopy structures are reported in which the full-length human α1β3γ2L GABA_A receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA, and the benzodiazepines alprazolam and diazepam.

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.

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.

The cystic fibrosis transmembrane conductance regulator anion channel can adopt an alternate conformation of its nucleotide-binding domain, which affects channel activity and, under certain conditions, leads to unfolding and protein degradation.

Cholesterol can function as both a substrate and an inhibitor of the Hedgehog receptor Patched. Structural analysis and molecular dynamics simulations reveal that cholesterol inhibits Patched by inserting into its extracellular domain

The active-state structure of a GPCR occupied by a partial agonist, β₂AR with salmeterol, together with mutagenesis and biophysical studies, explains this ligand's unusual pharmacological profile.

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.

A crystal structure of prokaryotic pentameric ligand-gated ion channel ELIC reveals a lipid-binding site at the interface between subunits that is shaped by a flexible helix important for channel desensitization upon lipid binding.

Mediator is a large multi-subunits complex essential to the regulation of transcription by RNA pol II. Here the authors report the crystal structure of MED23—one of the largest subunits of the complex together with MED1 and MED14—revealing a complex architecture and filling an important gap in the structural characterization of Mediator.

The X-ray crystal structure of the human β₂ adrenergic receptor, a G-protein-coupled receptor, in an agonist-bound 'active' state is solved. Comparison of this structure with a previously published structure of the same GPCR in an inactive state indicates that minor changes in the binding pocket of the protein lead to major changes elsewhere — there is a large outward movement of the cytoplasmic end of one of the transmembrane segments and rearrangements of two other transmembrane segments. This structure provides insights into the process of agonist binding and activation.

Stabilization of a transient protein kinase–substrate complex using a nanobody provides molecular details about how the Parkinson’s disease-linked protein kinase PINK1 phosphorylates ubiquitin, and suggests new pharmacological strategies.

Very little is known about how a G-protein-coupled receptor (GPCR) transitions from an inactive to an active state, but this study has solved the X-ray crystal structures of the human M2 muscarinic acetylcholine receptor bound to a high-affinity agonist in an active state and to a high-affinity agonist and a small-molecule allosteric modulator in an active state; the structures provide insights into the activation mechanism and allosteric modulation of muscarinic receptors.

G protein-coupled receptors (GPCRs) activate and dissociate the G protein heterotrimer into Gα-GTP and Gβγ dimer, which facilitate distinct signalling events. Here authors develop a nanobody, Nb5 that modulates Gβγ-mediated signaling without affecting GTP-bound Gαq and Gαs-mediated signaling events.

The X-ray crystal structure of ScaDMT, a bacterial member of the solute carrier 11 transporter family, identifies conserved residues within the substrate-binding site that confer metal-ion selectivity.

SLC26 membrane proteins constitute a large family of anion transporters with diverse functions. The first structure of a full-length SLC26 transporter now provides a common framework for the architecture of this protein family.

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.

A high-resolution cryo-electron microscopy structure is reported for the full-length human α1β3γ2L GABA_A receptor, functionally reconstituted in lipid nanodiscs.

Crystal structures of free and ligand-bound β-klotho reveal that it acts as a primary receptor for FGF21, and demonstrate how a sugar-cutting enzyme has evolved to become a receptor for hormones that regulate metabolic processes.

Structures of RNA polymerase of human and avian influenza A viruses reveal that the interface of the RNA polymerase dimer is required to initiate viral RNA synthesis in viral genome replication.

The use of nanobodies that inhibit the self-assembly of the S-layer protein Sap from B. anthracis enabled the elucidation of the structure of this protein. The nanobodies also trigger disintegration of assembled S-layers and attenuate both bacterial growth and anthrax pathology in animal models of infection.

Nanobody-aided X-ray crystallography and cryo-electron microscopy are used to describe the Ca²⁺-dependent polymerization dynamics of the S-layer of the Geobacillus stearothermophilus cell wall.

Stabilization of an active and inactive conformation of the β₂-adrenergic receptor by allosteric nanobodies reveals differential ligand-dependent regulation of receptor states to control G-protein-coupled receptor activation.

Here, pharmacological and biochemical evidence is provided that shows that G-protein coupling to the β₂-adrenergic receptor stabilizes a ‘closed’ conformation of the G-protein-coupled receptor (GPCR) and that that the effects of the G protein on the ligand-binding site of the GPCR are observed even in the absence of a bound agonist.