Mechanism of CFTR correction by type I folding correctors.

Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. To investigate the molecular mechanism of their action, we determined cryo-electron microscopy structures of CFTR in complex with the FDA-approved correctors lumacaftor or tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its premature degradation, and thereby allosterically rescuing many disease-causing mutations.

Universal recording of immune cell interactions in vivo.

Immune cells rely on transient physical interactions with other immune and non-immune populations to regulate their function1. To study these 'kiss-and-run' interactions directly in vivo, we previously developed LIPSTIC (labelling immune partnerships by SorTagging intercellular contacts)2, an approach that uses enzymatic transfer of a labelled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ T helper cells and antigen-presenting cells, however. Here we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the steady-state cellular partners of regulatory T cells and identify germinal centre-resident T follicular helper cells on the basis of their ability to interact cognately with germinal centre B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalogue of the immune populations that physically interact with intestinal epithelial cells at the steady state and profile the evolution of the interactome of lymphocytic choriomeningitis virus-specific CD8+ T cells in multiple organs following systemic infection. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.

Structures of Respiratory Supercomplex I+III2 Reveal Functional and Conformational Crosstalk.

The mitochondrial electron transport chain complexes are organized into supercomplexes (SCs) of defined stoichiometry, which have been proposed to regulate electron flux via substrate channeling. We demonstrate that CoQ trapping in the isolated SC I+III2 limits complex (C)I turnover, arguing against channeling. The SC structure, resolved at up to 3.8 Å in four distinct states, suggests that CoQ oxidation may be rate limiting because of unequal access of CoQ to the active sites of CIII2. CI shows a transition between "closed" and "open" conformations, accompanied by the striking rotation of a key transmembrane helix. Furthermore, the state of CI affects the conformational flexibility within CIII2, demonstrating crosstalk between the enzymes. CoQ was identified at only three of the four binding sites in CIII2, suggesting that interaction with CI disrupts CIII2 symmetry in a functionally relevant manner. Together, these observations indicate a more nuanced functional role for the SCs.

Structural basis for CFTR inhibition by CFTRinh-172.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. While activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease. Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTRinh-172 to an overall resolution of 2.7 Å by cryogenic electron microscopy. We observe that CFTRinh-172 binds inside the pore near transmembrane helix 8, a critical structural element that links adenosine triphosphate hydrolysis with channel gating. Binding of CFTRinh-172 stabilizes a conformation in which the chloride selectivity filter is collapsed, and the pore is blocked from the extracellular side of the membrane. Single-molecule fluorescence resonance energy transfer experiments indicate that CFTRinh-172 inhibits channel gating without compromising nucleotide-binding domain dimerization. Together, these data reconcile previous biophysical observations and provide a molecular basis for the activity of this widely used CFTR inhibitor.

The architecture of respiratory supercomplexes.

Mitochondrial electron transport chain complexes are organized into supercomplexes responsible for carrying out cellular respiration. Here we present three architectures of mammalian (ovine) supercomplexes determined by cryo-electron microscopy. We identify two distinct arrangements of supercomplex CICIII2CIV (the respirasome)-a major 'tight' form and a minor 'loose' form (resolved at the resolution of 5.8 Å and 6.7 Å, respectively), which may represent different stages in supercomplex assembly or disassembly. We have also determined an architecture of supercomplex CICIII2 at 7.8 Å resolution. All observed density can be attributed to the known 80 subunits of the individual complexes, including 132 transmembrane helices. The individual complexes form tight interactions that vary between the architectures, with complex IV subunit COX7a switching contact from complex III to complex I. The arrangement of active sites within the supercomplex may help control reactive oxygen species production. To our knowledge, these are the first complete architectures of the dominant, physiologically relevant state of the electron transport chain.

Atomic structure of the entire mammalian mitochondrial complex I.

Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) contributes to cellular energy production by transferring electrons from NADH to ubiquinone coupled to proton translocation across the membrane. It is the largest protein assembly of the respiratory chain with a total mass of 970 kilodaltons. Here we present a nearly complete atomic structure of ovine (Ovis aries) mitochondrial complex I at 3.9 Å resolution, solved by cryo-electron microscopy with cross-linking and mass-spectrometry mapping experiments. All 14 conserved core subunits and 31 mitochondria-specific supernumerary subunits are resolved within the L-shaped molecule. The hydrophilic matrix arm comprises flavin mononucleotide and 8 iron-sulfur clusters involved in electron transfer, and the membrane arm contains 78 transmembrane helices, mostly contributed by antiporter-like subunits involved in proton translocation. Supernumerary subunits form an interlinked, stabilizing shell around the conserved core. Tightly bound lipids (including cardiolipins) further stabilize interactions between the hydrophobic subunits. Subunits with possible regulatory roles contain additional cofactors, NADPH and two phosphopantetheine molecules, which are shown to be involved in inter-subunit interactions. We observe two different conformations of the complex, which may be related to the conformationally driven coupling mechanism and to the active-deactive transition of the enzyme. Our structure provides insight into the mechanism, assembly, maturation and dysfunction of mitochondrial complex I, and allows detailed molecular analysis of disease-causing mutations.

Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation.

NADH-ubiquinone oxidoreductase (complex I) is the largest (∼1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Because of the ease of sample availability, previous work has focused almost exclusively on bovine complex I. However, only medium resolution structural analyses of this complex have been reported. Working with other mammalian complex I homologues is a potential approach for overcoming these limitations. Due to the inherent difficulty of expressing large membrane protein complexes, screening of complex I homologues is limited to large mammals reared for human consumption. The high sequence identity among these available sources may preclude the benefits of screening. Here, we report the characterization of complex I purified from Ovis aries (ovine) heart mitochondria. All 44 unique subunits of the intact complex were identified by mass spectrometry. We identified differences in the subunit composition of subcomplexes of ovine complex I as compared with bovine, suggesting differential stability of inter-subunit interactions within the complex. Furthermore, the 42-kDa subunit, which is easily lost from the bovine enzyme, remains tightly bound to ovine complex I. Additionally, we developed a novel purification protocol for highly active and stable mitochondrial complex I using the branched-chain detergent lauryl maltose neopentyl glycol. Our data demonstrate that, although closely related, significant differences exist between the biochemical properties of complex I prepared from ovine and bovine mitochondria and that ovine complex I represents a suitable alternative target for further structural studies.

Universal recording of cell-cell contacts in vivo for interaction-based transcriptomics.

Cellular interactions are essential for tissue organization and functionality. In particular, immune cells rely on direct and usually transient interactions with other immune and non-immune populations to specify and regulate their function. To study these "kiss-and-run" interactions directly in vivo, we previously developed LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts), an approach that uses enzymatic transfer of a labeled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ helper T cells and antigen presenting cells, however. Here, we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the cellular partners of regulatory T cells in steady state, and identify germinal center (GC)-resident T follicular helper (Tfh) cells based on their ability to interact cognately with GC B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalog of the immune populations that physically interact with intestinal epithelial cells (IECs) and find evidence of stepwise acquisition of the ability to interact with IECs as CD4+ T cells adapt to residence in the intestinal tissue. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.

Molecular structures reveal synergistic rescue of Δ508 CFTR by Trikafta modulators.

The predominant mutation causing cystic fibrosis, a deletion of phenylalanine 508 (Δ508) in the cystic fibrosis transmembrane conductance regulator (CFTR), leads to severe defects in CFTR biogenesis and function. The advanced therapy Trikafta combines the folding corrector tezacaftor (VX-661), the channel potentiator ivacaftor (VX-770), and the dual-function modulator elexacaftor (VX-445). However, it is unclear how elexacaftor exerts its effects, in part because the structure of Δ508 CFTR is unknown. Here, we present cryo-electron microscopy structures of Δ508 CFTR in the absence and presence of CFTR modulators. When used alone, elexacaftor partially rectified interdomain assembly defects in Δ508 CFTR, but when combined with a type I corrector, did so fully. These data illustrate how the different modulators in Trikafta synergistically rescue Δ508 CFTR structure and function.

Mammalian Mitochondrial Complex I Structure and Disease-Causing Mutations.

Complex I has an essential role in ATP production by coupling electron transfer from NADH to quinone with translocation of protons across the inner mitochondrial membrane. Isolated complex I deficiency is a frequent cause of mitochondrial inherited diseases. Complex I has also been implicated in cancer, ageing, and neurodegenerative conditions. Until recently, the understanding of complex I deficiency on the molecular level was limited due to the lack of high-resolution structures of the enzyme. However, due to developments in single particle cryo-electron microscopy (cryo-EM), recent studies have reported nearly atomic resolution maps and models of mitochondrial complex I. These structures significantly add to our understanding of complex I mechanism and assembly. The disease-causing mutations are discussed here in their structural context.

Bactobolin A binds to a site on the 70S ribosome distinct from previously seen antibiotics.

The ribosome is the target of a large number of antibiotics. Here, we report a 3.4-Å-resolution crystal structure of bactobolin A bound to 70S ribosome-tRNA complex. The antibiotic binds at a previously unseen site in the 50S subunit and displaces tRNA bound at the P-site. It thus likely has a similar mechanism of action as blasticidin S despite binding to a different site. The structure also rationalizes previously identified resistance mutations.

Tubulin acetyltransferase αTAT1 destabilizes microtubules independently of its acetylation activity.

Acetylation of α-tubulin at lysine 40 (K40) is a well-conserved posttranslational modification that marks long-lived microtubules but has poorly understood functional significance. Recently, αTAT1, a member of the Gcn5-related N-acetyltransferase superfamily, has been identified as an α-tubulin acetyltransferase in ciliated organisms. Here, we explored the function of αTAT1 with the aim of understanding the consequences of αTAT1-mediated microtubule acetylation. We demonstrate that α-tubulin is the major target of αTAT1 but that αTAT1 also acetylates itself in a regulatory mechanism that is required for effective modification of tubulin. We further show that in mammalian cells, αTAT1 promotes microtubule destabilization and accelerates microtubule dynamics. Intriguingly, this effect persists in an αTAT1 mutant with no acetyltransferase activity, suggesting that interaction of αTAT1 with microtubules, rather than acetylation per se, is the critical factor regulating microtubule stability. Our data demonstrate that αTAT1 has cellular functions that extend beyond its classical enzymatic activity as an α-tubulin acetyltransferase.