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N -linked protein glycosylation is a post-translational modification that exists in all domains of life. It involves two consecutive steps: (i) biosynthesis of a lipid-linked oligosaccharide (LLO), and (ii) glycan transfer from the LLO to asparagine residues in secretory proteins, which is catalyzed by the integral membrane enzyme oligosaccharyltransferase (OST). In the last decade, structural and functional studies of the N -glycosylation machinery have increased our mechanistic understanding of the pathway. The structures of bacterial and eukaryotic glycosyltransferases involved in LLO elongation provided an insight into the mechanism of LLO biosynthesis, whereas structures of OST enzymes revealed the molecular basis of sequon recognition and catalysis. In this review, we will discuss approaches used and insight obtained from these studies with a special emphasis on the design and preparation of substrate analogs. [ABSTRACT FROM AUTHOR]

ABC transporters are essential for cellular physiology. Humans have 48 ABC genes organized into seven distinct families. Of these genes, 44 (in five distinct families) encode for membrane transporters, of which several are involved in drug resistance and disease pathways resulting from transporter dysfunction. Over the last decade, advances in structural biology have vastly expanded our mechanistic understanding of human ABC transporter function, revealing details of their molecular arrangement, regulation, and interactions, facilitated in large part by advances in cryo-EM that have rendered hitherto inaccessible targets amenable to high-resolution structural analysis. As a result, experimentally determined structures of multiple members of each of the five families of ABC transporters in humans are now available. Here we review this recent progress, highlighting the physiological relevance of human ABC transporters and mechanistic insights gleaned from their direct structure determination. We also discuss the impact and limitations of model systems and structure prediction methods in understanding human ABC transporters and discuss current challenges and future research directions. [ABSTRACT FROM AUTHOR]

Glycosyltransferases of the C superfamily (GT-Cs) are enzymes found in all domains of life. They catalyse the stepwise synthesis of oligosaccharides or the transfer of assembled glycans from lipid-linked donor substrates to acceptor proteins. The processes mediated by GT-Cs are required for C-, N- and O- linked glycosylation, all of which are essential post-translational modifications in higher-order eukaryotes. Until recently, GT-Cs were thought to share a conserved structural module of 7 transmembrane helices; however, recently determined GT-C structures revealed novel folds. Here we analyse the growing diversity of GT-C folds and discuss the emergence of two subclasses, termed GT-C A and GT-C B. Further substrate-bound structures are needed to facilitate a molecular understanding of glycan recognition and catalysis in these two subclasses. • C superfamily glycosyltransferases (GT-Cs) are present in all domains of life. • GT-Cs transfer sugars from lipid-linked donors to diverse acceptor substrates. • These enzymes have conserved catalytic and variable substrate binding modules. • New GT-C structures demonstrate at least two distinct folds of the catalytic module. • Cryo-EM has revolutionised structural characterisation of the GT-C superfamily. [ABSTRACT FROM AUTHOR]

Many animals change their body pigmentation according to illumination of their environment. In aquatic vertebrates, this reaction is mediated through aggregation or dispersion of melanin-filled vesicles (melanosomes) in dermal pigment cells (melanophores). The adaptive value of this behavior is usually seen in camouflage by allowing the animal to visually blend into the background. When exposed to visible light from below, however, dark-adapted zebrafish embryos at the age of 2 days post fertilization (dpf) surprisingly display dispersal instead of aggregation of melanosomes, i.e. their body coloration becomes dark on a bright background. Melanosomes of older embryos and early larvae (3–5 dpf) on the other hand aggregate as expected under these conditions. Here we provide an explanation to this puzzling finding: Melanosome dispersion in larvae 3 dpf and older is efficiently triggered by ultraviolet (UV) light, irrespective of the visual background, suggesting that the extent of pigmentation is a trade-off between threats from predation and UV irradiation. The UV light-induced dispersion of melanosomes thereby is dependent on input from retinal short wavelength-sensitive (SWS) cone photoreceptors. In young embryos still lacking a functional retina, protection from UV light predominates, and light triggers a dispersal of melanosomes via photoreceptors intrinsic to the melanophores, regardless of the actual UV content. In older embryos and early larvae with functional retinal photoreceptors in contrast, this light-induced dispersion is counteracted by a delayed aggregation in the absence of UV light. These data suggest that the primary function of melanosome dispersal has evolved as a protective adaption to prevent UV damage, which was only later co-opted for camouflage. [ABSTRACT FROM AUTHOR]

The research and utilization of the magnetic nanoparticles in, e.g., biology and medicine have been preferentially oriented on magnetite Fe3O4 and maghemite \gamma-Fe2O3. The complex oxides allow a better control of the magnetic properties in a desirable way. We demonstrate this approach on an example of the La1-xSr_xMnO3 perovskites from the point of view of their use in self-controlled magnetic fluid hyperthermia and increase of contrast in magnetic resonance imaging. With materials possessing suitable values of transition temperature TC, coercivity and magnetization, the heating power generated by the nanoparticles was estimated by two independent methods and they were finally employed in vitro and in vivo hyperthermia experiments. [ABSTRACT FROM AUTHOR]

Training experimental animals to discriminate between different visual stimuli has been an important tool in cognitive neuroscience as well as in vision research for many decades. Current methods used for visual choice discrimination training of zebrafish require human observers for response tracking, stimulus presentation and reward delivery and, consequently, are very labor intensive and possibly experimenter biased. By combining video tracking of fish positions, stimulus presentation on computer monitors and food delivery by computer-controlled electromagnetic valves, we developed a method that allows for a fully automated training of multiple adult zebrafish to arbitrary visual stimuli in parallel. The standardized training procedure facilitates the comparison of results across different experiments and laboratories and contributes to the usability of zebrafish as vertebrate model organisms in behavioral brain research and vision research. [ABSTRACT FROM AUTHOR]

Many arthropods including insects and spiders exploit skylight polarization for navigation. One of the four eye pairs of the spider Drassodes cupreus is dedicated to detect skylight polarization. These eyes are equipped with a tapetum that strongly plane-polarizes reflected light. This effectively enhances the polarization-sensitivity of the photoreceptors, improving orientation performance. With a multidisciplinary approach, we demonstrate that D. cupreus exploits reflective elements also present in non-polarizing tapetal eyes of other species such as Agelena labyrinthica. By approximately orthogonal arrangement of two multilayer reflectors consisting of reflecting guanine platelets, the tapetum uses the mechanism of polarization by reflection for polarizing reflected light. [ABSTRACT FROM AUTHOR]

Abstract: Small teleost fish are increasingly used for studying the genetic basis of vision. In particular, zebrafish (Danio rerio) and medaka (Oryzias latipes) are commonly used vertebrate model organisms in developmental research, including research on the development of visual function. A multitude of behavior-based visual tests are established for larvae that have been successfully used to identify and characterize visual defects in genetically manipulated strains of these species. Testing the visual system of adult fish has proven to be more difficult for a number of reasons, including complications in restraining fish, or shoaling and dominance behavior interfering with visual behavior in population screening assays. In this paper, we present a simple and cost-effective method to quantitatively measure the optokinetic response (OKR) of individual adult zebrafish and medaka, which can be used to characterize visual capabilities of adult fish. This method can be applied to any fish species of similar size. [Copyright &y& Elsevier]

ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins that includes both importers and exporters. In recent years, several structures of complete ABC transporters have been determined by X-ray crystallography. These structures suggest a mechanism by which binding and hydrolysis of ATP by the cytoplasmic, nucleotide-binding domains control the conformation of the transmembrane domains and therefore which side of the membrane the translocation pathway is exposed to. A basic, conserved two-state mechanism can explain active transport of both ABC importers and ABC exporters, but various questions remain unresolved. In this article, I will review some of the crystal structures and the mechanistic insight gained from them. Future challenges for a better understanding of the mechanism of ABC transporters will be outlined. [ABSTRACT FROM AUTHOR]

Abstract: Staphylococcus aureus Sav1866 is a bacterial homolog of the human ABC transporter Mdr1 that causes multidrug resistance in cancer cells. We report the crystal structure of Sav1866 in complex with adenosine-5′-(β,γ-imido)triphosphate (AMP-PNP) at 3.4Å resolution and compare it with the previously determined structure of Sav1866 with bound ADP. Besides differences in the ATP-binding sites, no significant conformational changes were observed. The results confirm that the ATP-bound state of multidrug ABC transporters is coupled to an outward-facing conformation of the transmembrane domains. [Copyright &y& Elsevier]

Multidrug transporters of the ABC family facilitate the export of diverse cytotoxic drugs across cell membranes. This is clinically relevant, as tumour cells may become resistant to agents used in chemotherapy. To understand the molecular basis of this process, we have determined the 3.0 Å crystal structure of a bacterial ABC transporter (Sav1866) from Staphylococcus aureus. The homodimeric protein consists of 12 transmembrane helices in an arrangement that is consistent with cross-linking studies and electron microscopic imaging of the human multidrug resistance protein MDR1, but critically different from that reported for the bacterial lipid flippase MsbA. The observed, outward-facing conformation reflects the ATP-bound state, with the two nucleotide-binding domains in close contact and the two transmembrane domains forming a central cavity—presumably the drug translocation pathway—that is shielded from the inner leaflet of the lipid bilayer and from the cytoplasm, but exposed to the outer leaflet and the extracellular space. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substrates across cell or organelle membranes. The recently determined crystal structures of the vitamin B12 importer BtuCD and its cognate binding protein BtuF have revealed critical architectural features that are probably shared by other ABC transporters. For example, the arrangement of the ABC domains and their interface with the membrane-spanning domains are probably conserved, whereas the number of transmembrane helices and their arrangement are not. Two distinct mechanistic schemes for how ABC engines couple ATP hydrolysis to substrate transport have been proposed recently and are being explored. [Copyright &y& Elsevier]

ABC transporters are ubiquitous membrane proteins that facilitate unidirectional substrate translocation across the lipid bilayer. Over the past five years, new crystal structures have advanced our understanding of how ABC transporters couple adenosine triphosphate (ATP) hydrolysis to substrate transport. In the following, we will briefly review the results of these structural investigations and outline their mechanistic implications. [Copyright &y& Elsevier]

The passage of most molecules across biological membranes is mediated by specialized integral membrane proteins known as channels and transporters. Although these transport families encompass a wide range of functions, molecular architectures and mechanisms, there are common elements that must be incorporated within their structures, namely the translocation pathway, ligand specificity elements and regulatory sensors to control the rate of ligand flow across the membrane. This minireview discusses aspects of the structure and mechanism of two bacterial transport systems, the stretch-activated mechanosensitive channel of small conductance (MscS) and the ATP-dependent vitamin B12 uptake system (BtuCD), emphasizing their general implications for transporter function. [Copyright &y& Elsevier]

Bacterial binding protein-dependent ATP binding cassette (ABC) transporters facilitate uptake of essential nutrients. The crystal structure of Escherichia coli BtuF, the protein that binds vitamin B[sub 12] and delivers it to the periplasmic surface of the ABC transporter BtuCD, reveals a bi-lobed fold resembling that of the ferrichrome binding protein FhuD. B[sub 12] is bound in the "base-on" conformation in a deep cleft formed at the interface between the two lobes of BtuF. A stable complex between BtuF and BtuCD (with the stoichiometry BtuC[sub 2]D[sub 2]F) is demonstrated to form in vitro and was modeled using the individual crystal structures. Two surface glutamates from BtuF may interact with arginine residues on the periplasmic surface of the BtuCD transporter. These glutamate and arginine residues are conserved among binding proteins and ABC transporters mediating iron and B[sub 12] uptake, suggesting that they may have a role in docking and the transmission of conformational changes. [ABSTRACT FROM AUTHOR]

The ABC transporters are ubiquitous membrane proteins that couple adenosine triphosphate (ATP) hydrolysis to the translocation of diverse substrates across cell membranes. Clinically relevant examples are associated with cystic fibrosis and with multidrug resistance of pathogenic bacteria and cancer cells. Here, we report the crystal structure at 3.2 angstrom resolution of the Escherichia coli BtuCD protein, an ABC transporter mediating vitamin B[sub 12] uptake. The two ATP-binding cassettes (BtuD) are in close contact with each other, as are the two membrane-spanning subunits (BtuC); this arrangement is distinct from that observed for the E. coli lipid flippase MsbA. The BtuC subunits provide 20 transmembrane helices grouped around a translocation pathway that is closed to the cytoplasm by a gate region whereas the dimer arrangement of the BtuD subunits resembles the ATP-bound form of the Rad50 DNA repair enzyme. A prominent cytoplasmic loop of BtuC forms the contact region with the ATP-binding cassette and appears to represent a conserved motif among the ABC transporters. [ABSTRACT FROM AUTHOR]

The outer membrane protects Gram-negative bacteria against a harsh environment. At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane α-helices, but instead fold into antiparallel β-barrels. Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined. They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA. These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins. Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them. [ABSTRACT FROM AUTHOR]

Provides information on a study which showed that FhuA protein facilitates ligand-gated transport of ferrichrome-bound iron across Escherichia coli outer membranes. Review of related literature; Overview of FhuA protein architecture; Binding site for ferrichrome; Discussion on signal transduction by local and allosteric transitions.

The channel-forming FhuA protein, which translocates ferrichrome across Escherichia coli outer membranes, binds 1 mol ligand/mol monomer in detergent solution. The protein is homogenous and migrates as a single band with a mobility corresponding to 77 kDa in SDS/PAGE electrophoresis. Analytical ultracentrifugation revealed a monodisperse species (s20,w = 3.8 S) with a mass of 77 800 ± 3 200 Da. The properties of ligand binding, determined by two independent methods, revealed one binding site/ monomer, but are complicated by a pronounced convexity of the Scatchard plot and a Hill coefficient calculated to be 2.5. This strongly suggests that oligomeric species are present. Cross-linking agents revealed the existence of possibly transient, mostly dimeric and trimeric species. The difference between the FhuA protein in detergent solution and in its native membrane environment may be related to the removal of lateral pressure that exists in situ. [ABSTRACT FROM AUTHOR]

We investigate the properties of a trace-gas sensing scheme based on Rydberg excitations at the example of an idealized model system. Rydberg states in thermal rubidium (Rb) are created using a 2-photon cw excitation. These Rydberg-excited atoms ionize via collisions with a background gas of nitrogen (N2). The emerging charges are then measured as an electric current, which is on the order of several picoampere. Due to the 2-photon excitation, this sensing method has a large intrinsic selectivity combined with a promising sensitivity of 10 ppb at an absolute concentration of 1 ppm. The determination of the detection limit is limited by the optical reference measurement but is at least 500 ppb. [ABSTRACT FROM AUTHOR]

Oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose glycan onto secretory proteins in the endoplasmic reticulum. Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner. Here, we present high-resolution cryo–electron microscopy structures of human OST-A and OST-B. Although they have similar overall architectures, structural differences in the catalytic subunits STT3A and STT3B facilitate contacts to distinct OST subunits, DC2 in OST-A and MAGT1 in OST-B. In OST-A, interactions with TMEM258 and STT3A allow ribophorin-I to form a four-helix bundle that can bind to a translating ribosome, whereas the equivalent region is disordered in OST-B. We observed an acceptor peptide and dolichylphosphate bound to STT3B, but only dolichylphosphate in STT3A, suggesting distinct affinities of the two OST complexes for protein substrates. [ABSTRACT FROM AUTHOR]

Planar line-defect photonic crystal waveguides with a weak vertical refractive index contrast (substrate-type) suffer from large propagation losses in the order of 600-1000 dB/cm. A possibility to reduce propagation losses is to optimise the vertical layer stack for a given hole shape. This strategy is pursued to reduce the propagation losses numerically and experimentally. It is demonstrated that the loss figure can vary by a factor of about 2 for relatively small changes in the core layer thickness. [ABSTRACT FROM AUTHOR]

Transporter proteins are integral membrane proteins that selectively mediate the passage of molecules across the otherwise impermeable barrier imposed by the phospholipid bilayer that surrounds all cells and organelles. The identification of more than 360 families of transporters through biochemical and genomic analyses highlights the importance of transport processes to cells. Transporter protein LacY mediates the coupled cotransport of lactose and H, whereas GlpT catalyzes the exchange of glycerol-3-phosphate for phosphate. The LacY and GlpT structures reveal that the expected 12 transmembrane helices in each protein are organized into two distinct domains composed of six helices in the amino terminus and six helices in the carboxyl terminus. The LacY and GlpT structures support the "alternating access" mechanism of transport in which the protein undergoes a series of conformational transitions such that the ligand-binding site is alternatively accessible to one side of the membrane or the other, but not to both sides simultaneously.

Bulk InGaAs layers were grown at 400 °C lattice-matched to InP semi-insulating substrates by molecular beam epitaxy. Si doping of the layers was performed by applying volume- and delta-doping techniques. The samples were characterized by capacitance-voltage, van der Pauw- Hall, secondary ion mass spectroscopy and photoluminescence measurements. Good agreement in terms of dependence of mobility and Burstein-Moss shift shift on doping concentration in samples doped by the two different techniques was obtained. Amphoteric behavior of Si was observed at doping concentrations higher than ~2.9×1019 cm-3 in both delta- and volume-doped samples. Degradation of InGaAs crystalline quality occurred in samples with Si concentrations higher than ~4×1019 cm-3. [ABSTRACT FROM AUTHOR]

Summary: Larval zebrafish lacking eyes and pineal organ show elevated activity levels and undirected light-seeking behaviour upon loss of illumination. This behaviour, termed dark photokinesis, is mediated by hypothalamic deep brain photoreceptors expressing melanopsin. [Copyright &y& Elsevier]

Large propagation losses in the order 600-1000 dB/cm were reported in the past for planar line-defect photonic crystal waveguides with a weak vertical refractive index contrast (substrate-type). Loss-relevant factors are a well-suited design of the photonic crystal pattern and a high-quality fabrication technology. Here, the focus is on the latter. Two methods-a thermally driven mass transport process and an ultra-slow, selective wet-etching process-are assessed with respect to their capability of enabling low-loss waveguides. With the resulting hole shape, we experimentally demonstrate record-low propagation losses of 154 dB/cm for W1 photonic crystal waveguides in the InP/InGaAsP system. [ABSTRACT FROM AUTHOR]

Summary: Orientation of animals towards or away from light is a simple behavior commonly found in the animal kingdom. A recent study using zebrafish larvae has revealed the underlying neural logic of this primal choice behavior, by differential use of the retinal ON- and OFF-pathways. [Copyright &y& Elsevier]

ABCG2 is an ATP-binding cassette (ABC) transporter that extrudes a wide range of xenobiotics and drugs from the cell and contributes to multidrug resistance in cancer cells. Following our recent structural characterization of topotecan-bound ABCG2, here, we present cryo-EM structures of ABCG2 under turnover conditions in complex with a special modulator and slow substrate, tariquidar, in nanodiscs. The structures reveal that similar to topotecan, tariquidar induces two distinct ABCG2 conformations under turnover conditions (turnover-1 and turnover-2). sscale molecular dynamics simulations of drug-bound and apo ABCG2 in native-like lipid bilayers, in both topotecan- and tariquidar-bound states, characterize the ligand size as a major determinant of its binding stability. The simulations highlight direct lipid-drug interactions for the smaller topotecan, which exhibits a highly dynamic binding mode. In contrast, the larger tariquidar occupies most of the available volume in the binding pocket, thus leaving little space for lipids to enter the cavity and interact with it. Similarly, when simulating ABCG2 in the apo inward-open state, we also observe spontaneous penetration of phospholipids into the binding cavity. The captured phospholipid diffusion pathway into ABCG2 offers a putative general path to recruit any hydrophobic/amphiphilic substrates directly from the membrane. Our simulations also reveal that ABCG2 rejects cholesterol as a substrate, which is omnipresent in plasma membranes that contain ABCG2. At the same time, cholesterol is found to prohibit the penetration of phospholipids into ABCG2. These molecular findings have direct functional ramifications on ABCG2's function as a transporter. [ABSTRACT FROM AUTHOR]

We present here the positional cloning of the Danio rerio albino mutant and show that the affected gene encodes Slc45a2. The human orthologous gene has previously been shown to be involved in human skin color variation, and mutations therein have been implicated in the disease OCA4. Through ultrastructural analysis of the melanosomes in albino alleles as well as the tyrosinase-deficient mutant sandy, we add new insights into the role of Slc45a2 in the production of melanin. To gain further understanding of the role of Slc45a2 and its possible interactions with other proteins involved in melanization, we further analyzed the role of the V- ATPase as a melanosomal acidifier. We show that it is possible to rescue the melanization potential of the albino melanosomes through genetic and chemical inhibition of V- ATPase, thereby increasing internal melanosome p H. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette (ABC) transporter BtuCD mediates the uptake of vitamin B12 across the inner membrane of Escherichia coli. Previous structures have shown the conformations of apo states, but the transport mechanism has remained unclear. Here we report the 3.5?Å crystal structure of the transporter-binding protein complex BtuCD-BtuF (BtuCD-F) trapped in an ?-?-imidoadenosine 5?-phosphate (AMP-PNP)-bound intermediate state. Although the ABC domains (BtuD subunits) form the expected closed sandwich dimer, the membrane-spanning BtuC subunits adopt a new conformation, with the central translocation pathway sealed by a previously unrecognized cytoplasmic gate. A fully enclosed cavity is thus formed approximately halfway across the membrane. It is large enough to accommodate a vitamin B12 molecule, and radioligand trapping showed that liposome-reconstituted BtuCD-F indeed contains bound B12 in the presence of AMP-PNP. In combination with engineered disulphide crosslinking and functional assays, our data suggest an unexpected peristaltic transport mechanism that is distinct from those observed in other ABC transporters. [ABSTRACT FROM AUTHOR]

Studies on membrane protein folding have focused on monomeric α-helical proteins and a major challenge is to extend this work to larger oligomeric membrane proteins. Here, we study the Escherichia coli (E. coli) ATP-binding cassette (ABC) transporter that imports vitamin B12 (the BtuCD protein) and use it as a model system for investigating the folding and assembly of a tetrameric membrane protein complex. Our work takes advantage of the modular organization of BtuCD, which consists of two transmembrane protein subunits, BtuC, and two cytoplasmically located nucleotide-binding protein subunits, BtuD. We show that the BtuCD transporter can be re-assembled from both prefolded and partly unfolded, urea denatured BtuC and BtuD subunits. The in vitro re-assembly leads to a BtuCD complex with the correct, native, BtuC and BtuD subunit stoichiometry. The highest rates of ATP hydrolysis were achieved for BtuCD re-assembled from partly unfolded subunits. This supports the idea of cooperative folding and assembly of the constituent protein subunits of the BtuCD transporter. BtuCD folding also provides an opportunity to investigate how a protein that contains both membrane-bound and aqueous subunits coordinates the folding requirements of the hydrophobic and hydrophilic subunits. [ABSTRACT FROM AUTHOR]

Mutations in ubiquitously expressed metabolic genes often lead to CNS-specific effects, presumably because of the high metabolic demands of neurons. However, mutations in omnipresent metabolic pathways can conceivably also result in cell type-specific effects because of cell-specific requirements for intermediate products. One such example is the zebrafish noir mutant, which we found to be mutated in the pdhb gene, coding for the E1 β subunit of the pyruvate dehydrogenase complex. This vision mutant is described as blind and was isolated because of its vision defect-related darker appearance. A detailed morphological, behavioral, and physiological analysis of the phenotype revealed an unexpected specific effect on the retina. Surprisingly, the cholinergic amacrine cells of the inner retina are affected earlier than the photoreceptors. This might be attributable to the inability of these cells to maintain production of their neurotransmitter acetylcholine. This is reflected in an earlier loss of motion vision, followed only later by a general loss of light perception. Since both characteristics of the phenotype are attributable to a loss of acetyl-CoA production by pyruvate dehydrogenase, we used a ketogenic diet to bypass this metabolic block and could indeed partially rescue vision and prolong survival of the larvae. The noir mutant provides a case for a systemic disease with ocular manifestation with a surprising specific effect on the retina given the ubiquitous requirement for the mutated gene. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a diverse array of substrates across cell membranes. We present here the dynamics of complex formation of three structurally characterized ABC transporters—the BtuCD vitamin B12 importer and MetNI d/l-methionine importer from Escherichia coli and the Hi1470/1 metal-chelate importer from Haemophilus influenzae—in complex with their cognate binding proteins. Similarly to other ABC importers, MetNI interacts with its binding protein with low affinity (Kd ∼10−4 M). In contrast, BtuCD–BtuF and Hi1470/1–Hi1472 form stable, high-affinity complexes (Kd ∼10−13 and 10−9 M, respectively). In BtuCD–BtuF, vitamin B12 accelerates the complex dissociation rate ∼107-fold, with ATP having an additional destabilizing effect. The findings presented here highlight substantial mechanistic differences between BtuCD–BtuF, and likely Hi1470/1–Hi1472, and the better-characterized maltose and related ABC transport systems, indicating that there is considerable mechanistic diversity within this large protein super-family. [ABSTRACT FROM AUTHOR]

Background: Visual acuity, the ability of the visual system to distinguish two separate objects at a given angular distance, is influenced by the optical and neuronal properties of the visual system. Although many factors may contribute, the ultimate limit is photoreceptor spacing. In general, at least one unstimulated photoreceptor flanked by two stimulated ones is needed to perceive two objects as separate. This critical interval is also referred to as the Nyquist frequency and is according to the Shannon sampling theorem the highest spatial frequency where a pattern can be faithfully transmitted. We measured visual acuity in a behavioral experiment and compared the data to the physical limit given by photoreceptor spacing in zebrafish larvae. Results: We determined visual acuity by using the optokinetic response (OKR), reflexive eye movements in response to whole field movements of the visual scene. By altering the spatial frequency we determined the visual acuity at approximately 0.16 cycles/degree (cpd) (minimum separable angle = 3.1°). On histological sections we measured the retinal magnification factor and the distance between double cones, that are thought to mediate motion perception. These measurements set the physical limit at 0.24 cpd (2.1°). Conclusion: The maximal spatial information as limited by photoreceptor spacing can not be fully utilized in a motion dependent visual behavior, arguing that the larval zebrafish visual system has not matured enough to optimally translate visual information into behavior. Nevertheless behavioral acuity is remarkable close to its maximal value, given the immature state of young zebrafish larvae. [ABSTRACT FROM AUTHOR]

Abstract: BtuCD is a type II ABC importer that catalyzes the translocation of vitamin B12 from the periplasm into the cytoplasm of Escherichia coli. Crystal structures of BtuCD and the related HiF (or Hi1470/71) protein from Haemophilus influenzae have revealed distinct conformations of the transmembrane domains that form inner and outer gates. We used electron spin resonance spectroscopy to study the reaction cycle of BtuCD after labeling the protein at residues located at these gates. The results suggest that BtuCD as a prototype type II ABC importer may have a mechanism that is distinct from that of ABC exporters such as Sav1866 or type I ABC importers such as those specific for molybdate (ModBC) or maltose (MalFGK). Structured summary: MINT-6803800: btuF (uniprotkb:P37028), btuC (uniprotkb:P06609) and btuD (uniprotkb:P06611) physically interact (MI:0218) by molecular sieving (MI:0071) [Copyright &y& Elsevier]

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins that couple the transport of diverse substrates across cellular membranes to the hydrolysis of ATP. The crystal structures of four ABC transporters have recently been determined. They reveal similar arrangements of the conserved ATP-hydrolyzing nucleotide-binding domains, but unrelated architectures of the transmembrane domains, with the notable exception of a common ‘coupling helix’ that is essential for transmitting conformational changes. The structures suggest a mechanism that rationalizes ATP-driven transport: While binding of ATP appears to trigger an outward-facing conformation, dissociation of the hydrolysis products may promote an inward-facing conformation. This basic scheme can, in principle, explain nutrient import by ABC importers and drug extrusion by ABC exporters. [Copyright &y& Elsevier]

ATP-binding cassette (ABC) transporters are integral membrane proteins that move diverse substrates across cellular membranes. ABC importers catalyse the uptake of essential nutrients from the environment, whereas ABC exporters facilitate the extrusion of various compounds, including drugs and antibiotics, from the cytoplasm. How ABC transporters couple ATP hydrolysis to the transport reaction has long remained unclear. The recent crystal structures of four complete ABC transporters suggest that a key step of the molecular mechanism is conserved in importers and exporters. Whereas binding of ATP promotes an outward-facing conformation, the release of the hydrolysis products ADP and phosphate promotes an inward-facing conformation. This basic scheme can in principle explain ATP-driven drug export and binding protein-dependent nutrient uptake. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporter proteins carry diverse substrates across cell membranes. Whereas clinically relevant ABC exporters are implicated in various diseases or cause multidrug resistance of cancer cells, bacterial ABC importers are essential for the uptake of nutrients, including rare elements such as molybdenum. A detailed understanding of their mechanisms requires direct visualization at high resolution and in distinct conformations. Our recent structure of the multidrug ABC exporter Sav1866 has revealed an outward-facing conformation of the transmembrane domains coupled to a closed conformation of the nucleotide-binding domains, reflecting the ATP-bound state. Here we present the 3.1 Å crystal structure of a putative molybdate transporter (ModB2C2) from Archaeoglobus fulgidus in complex with its binding protein (ModA). Twelve transmembrane helices of the ModB subunits provide an inward-facing conformation, with a closed gate near the external membrane boundary. The ATP-hydrolysing ModC subunits reveal a nucleotide-free, open conformation, whereas the attached binding protein aligns the substrate-binding cleft with the entrance to the presumed translocation pathway. Structural comparison of ModB2C2A with Sav1866 suggests a common alternating access and release mechanism, with binding of ATP promoting an outward-facing conformation and dissociation of the hydrolysis products promoting an inward-facing conformation. [ABSTRACT FROM AUTHOR]

Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR, encoded by the CD320 gene. Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo‐TC (TC in complex with B12) or the interface of the human holo‐TC:TCblR complex with nanomolar affinities. We determined X‐ray crystal structures of these nanobodies bound to holo‐TC:TCblR, which enabled us to map their binding epitopes. When conjugated to the model toxin saporin, three of our nanobodies caused growth inhibition of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TC‐Nb4) using fluorescent light microscopy. The co‐crystal structure of holo‐TC:TCblR with another nanobody (TC‐Nb34) revealed novel features of the interface of TC and the LDLR‐A1 domain of TCblR, rationalizing the decrease in the affinity of TC‐B12 binding caused by the Δ88 mutation in CD320. [ABSTRACT FROM AUTHOR]

The oligosaccharyltransferase (OST) is the central enzyme in the N -glycosylation pathway. It transfers a defined oligosaccharide from a lipid-linker onto the asparagine side chain of proteins. The yeast OST consists of eight subunits and exists in two catalytically distinct isoforms that differ in one subunit, Ost3p or Ost6p. The cryo-electron microscopy structure of the Ost6p containing complex was found to be highly similar to the Ost3p containing OST. OST enzymes with altered Ost3p/Ost6p subunits were generated and functionally analyzed. The three C-terminal transmembrane helices were responsible for the higher turnover-rate of the Ost3p vs. the Ost6p containing enzyme in vitro and the more severe hypoglycosylation in Ost3p lacking strains in vivo. Glycosylation of specific OST target sites required the N-terminal thioredoxin domain of Ost3p or Ost6p. This Ost3p/Ost6p dependence was glycosylation site but not protein specific. We concluded that the Ost3p/Ost6p subunits modulate the catalytic activity of OST and provide additional specificity for OST substrate recognition. [ABSTRACT FROM AUTHOR]

[Display omitted] • ABCG1 transports cholesterol as part of reverse cholesterol transport pathway. • Cryo-EM structure of human ABCG1 in an inward open conformation. • Comparison with ABCG2 reveals basis for substrate specificity differences. • First insight into ABCG1-mediated cholesterol recognition. ABCG1 is an ATP binding cassette (ABC) transporter that removes excess cholesterol from peripheral tissues. Despite its role in preventing lipid accumulation and the development of cardiovascular and metabolic disease, the mechanism underpinning ABCG1-mediated cholesterol transport is unknown. Here we report a cryo-EM structure of human ABCG1 at 4 Å resolution in an inward-open state, featuring sterol-like density in the binding cavity. Structural comparison with the multidrug transporter ABCG2 and the sterol transporter ABCG5/G8 reveals the basis of mechanistic differences and distinct substrate specificity. Benzamil and taurocholate inhibited the ATPase activity of liposome-reconstituted ABCG1, whereas the ABCG2 inhibitor Ko143 did not. Based on the structural insights into ABCG1, we propose a mechanism for ABCG1-mediated cholesterol transport. [ABSTRACT FROM AUTHOR]

With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here, we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon complementary-determining region grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the highresolution cryogenic electron microscopy (cryo-EM) structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ~50 kDa. Using an additional anti-Fab nanobody further facilitated reliable initial three-dimensional structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, is humanized, and can be conveniently expressed in Escherichia coli in large amounts, it may be useful not only for structural biology but also for biomedical applications. [ABSTRACT FROM AUTHOR]

ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate the cytotoxicity of bile salts, is poorly understood. We present cryogenic electron microscopy structures of human ABCB4 in three distinct functional conformations. An apo-inward structure reveals how phospholipid can be recruited from the inner leaflet of the membrane without flipping its orientation. An occluded structure reveals a single phospholipid molecule in a central cavity. Its choline moiety is stabilized by cation-π interactions with an essential tryptophan residue, rationalizing the specificity of ABCB4 for phosphatidylcholine. In an inhibitor-bound structure, a posaconazole molecule blocks phospholipids from reaching the central cavity. Using a proteoliposome-based translocation assay with fluorescently labeled phosphatidylcholine analogs, we recapitulated the substrate specificity of ABCB4 in vitro and confirmed the role of the key tryptophan residue. Our results provide a structural basis for understanding an essential translocation step in the generation of bile and its sensitivity to azole drugs. [ABSTRACT FROM AUTHOR]

Transport proteins are essential for cells in allowing the exchange of substances between cells and their environment across the lipid bilayer forming a tight barrier. Membrane lipids modulate the function of transmembrane proteins such as transporters in two ways: Lipids are tightly and specifically bound to transport proteins and in addition they modulate from the bulk of the lipid bilayer the function of transport proteins. This overview summarizes currently available information at the ultrastructural level on lipids tightly bound to transport proteins and the impact of altered bulk membrane lipid composition. Human diseases leading to altered lipid homeostasis will lead to altered membrane lipid composition, which in turn affect the function of transporter proteins. • Transport proteins are key in allowing the passage of solutes across tight cellular lipid membrane barriers. • The activity of transport proteins is modulated among others by membrane lipids. • Membrane lipids modulate the activity of transport proteins by annular (lipids tightly bound to transporters) or membrane bulk lipids. • Cryo-EM technology yields a rapidly expanding list of transport protein structures with information on annular lipids. [ABSTRACT FROM AUTHOR]

GMI effect in permalloy can be used to measure DC magnetic fields. Impedance change of 50% at a field of 0.5 mT was observed at 200 kHz frequency. Magnetometer using biased two-stripe 10-cm long sensor achieved 10% linearity in open loop and 1% hysteresis error in the ±80 μT range. [Copyright &y& Elsevier]

Digital hysteresisgraph can be used for the measurement of dynamic hysteresis loops up to 100 kHz. Digital feedback allows achievement of sinusoidal flux density by iterative modification of the excitation voltage waveform. This approach is used for the measurement of closed (toroidal) samples at higher frequencies. [Copyright &y& Elsevier]

Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits. [ABSTRACT FROM AUTHOR]

ABCB1 detoxifies cells by exporting diverse xenobiotic compounds, thereby limiting drug disposition and contributing to multidrug resistance in cancer cells. Multiple small-molecule inhibitors and inhibitory antibodies have been developed for therapeutic applications, but the structural basis of their activity is insufficiently understood. We determined cryo-EM structures of nanodiscreconstituted, human ABCB1 in complex with the Fab fragment of the inhibitory, monoclonal antibody MRK16 and bound to a substrate (the antitumor drug vincristine) or to the potent inhibitors elacridar, tariquidar, or zosuquidar. We found that inhibitors bound in pairs, with one molecule lodged in the central drug-binding pocket and a second extending into a phenylalanine-rich cavity that we termed the “access tunnel.” This finding explains how inhibitors can act as substrates at low concentration, but interfere with the early steps of the peristaltic extrusion mechanism at higher concentration. Our structural data will also help the development of more potent and selective ABCB1 inhibitors. [ABSTRACT FROM AUTHOR]

Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N -glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo , but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc 2 -Man 9 -Glc 3 , suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action. [Display omitted] • Loss of OST-A subunit STT3A, but not its paralog STT3B, blocks LPS signaling to NF-κB • STT3A is required for N -glycosylation and membrane localization of the LPS receptor TLR4 • CRISPR base editing and cryo-EM defined a drug-binding pocket in STT3A • NGI-1 prevents a productive binding pose of the glycan donor in the active site of STT3A Innovative approaches identify druggable glycosylation processes that regulate TLR4-mediated activation of the pro-inflammatory transcription factor NF-κB. [ABSTRACT FROM AUTHOR]