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The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes 1 . Alterations in CFTR cause cystic fibrosis, a fatal disease without a cure 2 , 3 . Electrophysiological properties of CFTR have been analysed for decades 4 – 6 . The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology and kinetic simulations to show that the two nucleotide-binding domains (NBDs) of human CFTR dimerize before channel opening. CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while NBDs are dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization. These findings collectively enable the framing of a gating mechanism that informs on the search for more efficacious clinical therapies. A structure–function analysis of cystic fibrosis transmembrane conductance regulator shows its two nucleotide-binding domains dimerize before channel opening, and reveals a mechanism through which conformational changes in the channel regulate chloride conductance.

Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-Å resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6′-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding.

G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β 2 adrenergic receptor (β 2 AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the G s heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β 2 AR–G s complexes are formed and the efficiency of nucleotide exchange leading to G s activation. These data also reveal transient nucleotide-bound β 2 AR–G s species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism. Single-molecule FRET imaging provides insights into the allosteric link between the ligand-binding and G-protein nucleotide-binding pockets of the β 2 adrenergic receptor (β 2 AR) and improved understanding of the G-protein activation mechanism. Monitoring G-protein activation by a GPCR G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease, and understanding the molecular basis of ligand efficacy downstream of receptor activation is important for therapeutic development. For the GPCR β 2 adrenergic receptor (β 2 AR), receptor activation and coupling to the G protein G s involve outward movements of the receptor transmembrane helix 6 (TM6). Here, Scott Blanchard and colleagues apply single-molecule fluorescence resonance energy transfer (smFRET) imaging methods to directly monitor movements of TM6 in β 2 AR bound to a range of ligands with distinct efficacy profiles. They find that partial and full agonists affect TM6 motions in an efficacy-dependent manner. These motions differentially regulate the rate at which β 2 AR couples with GDP-bound G s and the efficiency of nucleotide exchange leading to G s activation. The work provides single-molecule insight into the allosteric link between the ligand- and G-protein-nucleotide-binding pockets of the receptor and improved understanding of the G-protein activation mechanism.

Single-molecule fluorescence resonance energy transfer imaging of a bacterial glutamate transporter reveals how the transport domains move. Glutamate transport dissected Glutamate transporters have a central role in neural transmission by maintaining low concentrations of neurotransmitter within synapses in the brain. The mechanism of transport involves transitions between extracellular- and intracellular-facing conformations, whereby substrate-binding sites become accessible to the opposite sides of the plasma membrane. Two papers in this issue of Nature report the use of single-molecule fluorescence resonance energy transfer imaging to directly observe large-scale transport domain movements in bacterial/archaeal homologues of glutamate transporters. Nurunisa Akyuz et al . find that individual transport domains alternate between periods of quiescence (reflecting stable conformations that closely resemble crystal structures of the outward- and inward-facing states) and periods of rapid transition (reflecting transmembrane movements of individual transport domains). Guus Erkens et al . show that the three Glt Ph subunits undergo conformational changes stochastically, and independently of each other. Glutamate transporters are integral membrane proteins that catalyse neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons 1 . Their mechanism of action involves transitions between extracellular (outward)-facing and intracellular (inward)-facing conformations, whereby substrate binding sites become accessible to either side of the membrane 2 . This process has been proposed to entail transmembrane movements of three discrete transport domains within a trimeric scaffold 3 . Using single-molecule fluorescence resonance energy transfer (smFRET) imaging 4 , we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods 5 , 6 . We propose that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent transmembrane movements and may be rate determining in the transport cycle.

Peptide-chain elongation during protein synthesis entails sequential aminoacyl-tRNA selection and translocation reactions that proceed rapidly (2–20 per second) and with a low error rate (around 10 −3  to 10 −5 at each step) over thousands of cycles 1 . The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life. Here we use single-molecule fluorescence methods to guide the capture of structures of early translocation events on the bacterial ribosome. Our findings reveal that the bacterial GTPase elongation factor G specifically engages spontaneously achieved ribosome conformations while in an active, GTP-bound conformation to unlock and initiate peptidyl-tRNA translocation. These findings suggest that processes intrinsic to the pre-translocation ribosome complex can regulate the rate of protein synthesis, and that energy expenditure is used later in the translocation mechanism than previously proposed. Cryo-electron microscopy and single-molecule fluorescence methods are used to elucidate the mechanism of early translocation events on the bacterial ribosome.

The HIV-1 envelope (Env) mediates viral entry into host cells. To enable the direct imaging of conformational dynamics within Env, we introduced fluorophores into variable regions of the glycoprotein gp120 subunit and measured single-molecule fluorescence resonance energy transfer within the context of native trimers on the surface of HIV-1 virions. Our observations revealed unliganded HIV-1 Env to be intrinsically dynamic, transitioning between three distinct prefusion conformations, whose relative occupancies were remodeled by receptor CD4 and antibody binding. The distinct properties of neutralization-sensitive and neutralization-resistant HIV-1 isolates support a dynamics-based mechanism of immune evasion and ligand recognition.

High-resolution structure of the E. coli ribosome highlights rRNA and protein modifications and provides details on solvation characteristics and the structural impacts of ribosome modifications. Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation have remained poorly understood. Moreover, the functions of modifications to ribosomal RNA and ribosomal proteins have also been unclear. Here we present the structure of the Escherichia coli 70S ribosome at 2.4-Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and it suggests how solvation contributes to ribosome integrity and function as well as how the conformation of ribosomal protein uS12 aids in mRNA decoding. This structure helps to explain the phylogenetic conservation of key elements of the ribosome, including post-transcriptional and post-translational modifications, and should serve as a basis for future antibiotic development.

Monitoring ribosomal translocation from five structural perspectives with pre–steady state smFRET defines intramolecular conformational events within the EF-G–GDP–bound ribosome as rate-determining steps of directional substrate translocation. Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre–steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G–GDP–bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.

The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. Here, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showed that the N-terminal portion of the fusion peptide can be solvent-exposed. These results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.

Accurate protein synthesis is determined by the two-subunit ribosome’s capacity to selectively incorporate cognate aminoacyl-tRNA for each mRNA codon. The molecular basis of tRNA selection accuracy, and how fidelity can be affected by antibiotics, remains incompletely understood. Using molecular simulations, we find that cognate and near-cognate tRNAs delivered to the ribosome by Elongation Factor Tu (EF-Tu) can follow divergent pathways of motion into the ribosome during both initial selection and proofreading. Consequently, cognate aa-tRNAs follow pathways aligned with the catalytic GTPase and peptidyltransferase centers of the large subunit, while near-cognate aa-tRNAs follow pathways that are misaligned. These findings suggest that differences in mRNA codon-tRNA anticodon interactions within the small subunit decoding center, where codon-anticodon interactions occur, are geometrically amplified over distance, as a result of this site’s physical separation from the large ribosomal subunit catalytic centers. These insights posit that the physical size of both tRNA and ribosome are key determinants of the tRNA selection fidelity mechanism. Protein synthesis is dependent on the ribosome’s ability to accurately select tRNA. Molecular simulations reveal divergent pathways for correct and incorrect tRNA during selection, indicating that tRNA alignment is key to protein production.