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The T-cell antigen receptor (TCR) is an assembly of eight type I single-pass membrane proteins that occupies a central position in adaptive immunity. Many TCR-triggering models invoke an alteration in receptor complex structure as the initiating event, but both the precise subunit organization and the pathway by which ligand-induced alterations are transferred to the cytoplasmic signaling domains are unknown. Here, we show that the receptor complex transmembrane (TM) domains form an intimately associated eight-helix bundle organized by a specific interhelical TCR TM interface. The salient features of this core structure are absolutely conserved between αβ and γδ TCR sequences and throughout vertebrate evolution, and mutations at key interface residues caused defects in the formation of stable TCRαβ:CD3δε:CD3γε:ζζ complexes. These findings demonstrate that the eight TCR–CD3 subunits form a compact and precisely organized structure within the membrane and provide a structural basis for further investigation of conformationally regulated models of transbilayer TCR signaling.

Glucose and fructose play a central role in the metabolism and cellular homeostasis of organisms. Their absorption is co-mediated by two families of glucose transporters, Na+-coupled glucose co-transporters (SGLTs) and facilitative Na+-independent sugar carriers (GLUTs), in the intestine. However, limited information has been available on these transporters in fish. Therefore, we studied glut2, sglt1, and sglt4 genes in grass carp (Ctenopharyngodon idellus). The full-length cDNAs of glut2 was 2308 bp, with an open reading frame (ORF) of 503 amino acids (AAs). The full-length cDNAs of sglt1 was 2890 bp, with an ORF of 658 AAs. Additionally, the full-length cDNAs of sglt4 was 2090 bp, with an ORF encoding 659 AAs. The three deduced AA sequences showed high homology between grass carp and other cyprinid fish species. Based on homology modeling, three-dimensional models of GLUT2, SGLT1, and SGLT4 proteins were created and transmembrane domains were noted. glut2, sglt1, and sglt4 were abundantly expressed in the anterior and mid intestine. In particular, glut2 was markedly expressed in liver (P < 0.05). Additionally, the results indicated that different stocking densities (0.9 or 5.9 kg m−2) did not alter intestinal section-dependent expression patterns of the three transporter genes. However, high stocking density impacted segmental mRNA expression levels. This work demonstrated that mRNA expression of sugar transporter genes in the fish intestine was segment specific, and crowding stress may affect the activity of intestinal sugar transporters. These results provided new insights into the relationship between crowding stress and intestinal sugar transporters in fish.

Purpose The thyroid-stimulating hormone receptor (TSHR) is the target autoantigen for TSHR-stimulating autoantibodies in Graves’ disease. The TSHR is composed of: a leucine-rich repeat domain (LRD), a hinge region or cleavage domain (CD) and a transmembrane domain (TMD). The binding arrangements between the TSHR LRD and the thyroid-stimulating autoantibody M22 or TSH have become available from the crystal structure of the TSHR LRD–M22 complex and a comparative model of the TSHR LRD in complex with TSH, respectively. However, the mechanism by which the TMD of the TSHR and the other glycoprotein hormone receptors (GPHRs) becomes activated is unknown. Methods We have generated comparative models of the structures of the inactive (TMD_In) and active (TMD_Ac) conformations of the TSHR, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) TMDs. The structures of TMD_Ac and TMD_In were obtained using class A GPCR crystal structures for which fully active and inactive conformations were available. Results Most conserved motifs observed in GPCR TMDs are also observed in the amino acid sequences of GPHR TMDs. Furthermore, most GPCR TMD conserved helix distortions are observed in our models of the structures of GPHR TMDs. Analysis of these structures has allowed us to propose a mechanism for activation of GPHR TMDs. Conclusions Insight into the mechanism of activation of the TSHR by both TSH and TSHR autoantibodies is likely to be useful in the development of new treatments for Graves’ disease.

Abstract FtsY is the receptor of the signal recognition particle that mediates the targeting of integral membrane proteins in bacteria. It was shown that in Escherichia coli, the N-terminal region of FtsY contributes to its interaction with the membrane, but it is not inserted into the membrane. However, this study presents evidence that in Streptomyces coelicolor, FtsY has a hydrophobic region at its N-terminus, which forms a membrane insertion structure and contributes significantly to the binding between FtsY and membrane. Through membrane protein extraction followed by immunoblotting, we demonstrated that deletion of the N-terminal residues 11–39 from the S. coelicolor FtsY (ScFtsY) drastically reduced its membrane-binding capability and that the N-terminus of ScFtsY alone was capable of targeting the soluble EGFP protein onto the membrane with high efficiency. Furthermore, in a labeling experiment with the membrane-impermeable probe Mal-PEG, the ScFtsY N-terminal region was protected by the membrane and was not labeled. This observation indicates that this region was inserted into the membrane.

This chapter contains sections titled: Introduction Comparative Modeling Experimental Data Fitting Quality Assessment Acknowledgments References

Cystic fibrosis transmembrane conductance regulator (CFTR), involved in cystic fibrosis (CF), is a chloride channel belonging to the ATP-binding cassette (ABC) superfamily. Using the experimental structure of Sav1866 as template, we previously modeled the human CFTR structure, including membrane-spanning domains (MSD) and nucleotide-binding domains (NBD), in an outward-facing conformation (open channel state). Here, we constructed a model of the CFTR inward-facing conformation (closed channel) on the basis of the recent corrected structures of MsbA and compared the structural features of those two states of the channel. Interestingly, the MSD:NBD coupling interfaces including F508 (ΔF508 being the most common CF mutation) are mainly left unchanged. This prediction, completed by the modeling of the regulatory R domain, is supported by experimental data and provides a molecular basis for a better understanding of the functioning of CFTR, especially of the structural features that make CFTR the unique channel among the ABC transporters.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel important in maintaining proper functions of the lung, pancreas, and intestine. The activity of CFTR is regulated by ATP and protein kinase A-dependent phosphorylation. To understand the conformational changes elicited by phosphorylation and ATP binding, we present here the structure of phosphorylated, ATP-bound human CFTR, determined by cryoelectronmicroscopy to 3.2-Å resolution. This structure reveals the position of the R domain after phosphorylation. By comparing the structures of human CFTR and zebrafish CFTR determined under the same condition, we identified common features essential to channel gating. The differences in their structures indicate plasticity permitted in evolution to achieve the same function. Finally, the structure of CFTR provides a better understanding of why the G178R, R352Q, L927P, and G970R/D mutations would impede conformational changes of CFTR and lead to cystic fibrosis.

Available corrector drugs are unable to effectively rescue the folding defects of CFTR-ΔF508 (or CFTR-F508del), the most common disease-causing mutation of the cystic fibrosis transmembrane conductance regulator, a plasma membrane (PM) anion channel, and thus to substantially ameliorate clinical phenotypes of cystic fibrosis (CF). To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutant expression and function at the PM. High-throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at nucleotide-binding domain (NBD1), NBD2 and their membrane-spanning domain (MSD) interfaces. Although individually these compounds marginally improve ΔF508-CFTR folding efficiency, function and stability, their combinations lead to ~50–100% of wild-type-level correction in immortalized and primary human airway epithelia and in mouse nasal epithelia. Likewise, corrector combinations were effective against rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application. Targeting different aspects of mutant CFTR structural defects with combination therapy leads to more potent rescue of function than that following single therapy.

Loss-of-function mutations of the CFTR gene cause the life-shortening genetic disease cystic fibrosis (CF), whereas overactivity of CFTR may lead to secretory diarrhea and polycystic kidney disease. While effective drugs targeting the CFTR protein have been developed for the treatment of CF, little progress has been made for diseases caused by hyper-activated CFTR. Here, we solve the cryo-EM structure of CFTR in complex with CFTRinh-172 (Inh-172), a CFTR gating inhibitor with promising potency and efficacy. We find that Inh-172 binds inside the pore of CFTR, interacting with amino acid residues from transmembrane segments (TMs) 1, 6, 8, 9, and 12 through mostly hydrophobic interactions and a salt bridge. Substitution of these residues lowers the apparent affinity of Inh-172. The inhibitor-bound structure reveals re-orientations of the extracellular segment of TMs 1, 8, and 12, supporting an allosteric modulation mechanism involving post-binding conformational changes. This allosteric inhibitory mechanism readily explains our observations that pig CFTR, which preserves all the amino acid residues involved in Inh-172 binding, exhibits a much-reduced sensitivity to Inh-172 and that the apparent affinity of Inh-172 is altered by the CF drug ivacaftor (i.e., VX-770) which enhances CFTR’s activity through binding to a site also comprising TM8. Using cryogenic electron microscopy, Gao et al. uncovered how CFTRInh-172 inhibits CFTR function by binding in its pore and allosterically inhibiting its gating. Their findings could pave the way for structure-based drug design for the treatment of secretory diarrhea and polycystic kidney disease.

TDP-43 regulates alternative splicing of the cystic fibrosis transmembrane regulator (CFTR) and is found in cytosolic granules associated with several neurodegenerative disorders. A new solution structure of the tandem RNA-recognition motifs (RRMs) that mediate interactions with its UG-rich RNA targets reveals a new RRM arrangement critical for TDP-43 function. TDP-43 encodes an alternative-splicing regulator with tandem RNA-recognition motifs (RRMs). The protein regulates cystic fibrosis transmembrane regulator ( CFTR ) exon 9 splicing through binding to long UG-rich RNA sequences and is found in cytoplasmic inclusions of several neurodegenerative diseases. We solved the solution structure of the TDP-43 RRMs in complex with UG-rich RNA. Ten nucleotides are bound by both RRMs, and six are recognized sequence specifically. Among these, a central G interacts with both RRMs and stabilizes a new tandem RRM arrangement. Mutations that eliminate recognition of this key nucleotide or crucial inter-RRM interactions disrupt RNA binding and TDP-43–dependent splicing regulation. In contrast, point mutations that affect base-specific recognition in either RRM have weaker effects. Our findings reveal not only how TDP-43 recognizes UG repeats but also how RNA binding–dependent inter-RRM interactions are crucial for TDP-43 function.