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Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl-CoAs (1-4). Because the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologues (SCD1 and SCD5), while mice have four (SCD1-SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity (5, 6). Thus, SCD1 is a pharmacological target in the treatment of obesity, diabetes and other metabolic diseases (7). SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyses the formation of a cis-double bond between the ninth and tenth carbons of stearoyl- or palmitoyl-CoA (8, 9). The reaction requires molecular oxygen, which is activated by a di-iron centre, and cytochrome [b.sub.5], which regenerates the di-iron centre (10). To understand better the structural basis of these characteristics of SCD function, here we crystallize and solve the structure of mouse SCD1 bound to stearoyl-CoA at 2.6 Å resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain, and a plausible pathway for lateral substrate access and product egress. The acyl chain of the bound stearoyl-CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel and the conformation of the bound acyl chain provide a structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The dimetal centre is coordinated by a unique spacial arrangement of nine conserved histidine residues that implies a potentially novel mechanism for oxygen activation. The structure also illustrates a possible route for electron transfer from cytochrome [b.sub.5] to the di-iron centre., A Δ2-23 amino-terminal truncation of mouse SCD1 was crystallized in lipidic cubic phase (11) and the structure was solved by single-wavelength anomalous dispersion (Extended Data Table 1). SCD1 has four [...]

Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the small intestine, where they aid in absorption of lipids and fat-soluble vitamins. Through a process known as enterohepatic recirculation, more than 90% of secreted bile acids are then retrieved from the intestine and returned to the liver for resecretion (1). In humans, there are two [Na.sup.+]-dependent bile acid transporters involved in enterohepatic recirculation, the [Na.sup.+]-taurocholate co-transporting polypeptide (NTCP; also known as SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT; also known as SLC10A2) expressed on enterocytes in the terminal ileum (2). In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolaemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption (3,4). However, a lack of three-dimensional structures of bile acid transporters hampers our ability to understand the molecular mechanisms of substrate selectivity and transport, and to interpret the wealth of existing functional data (2,5-8). The crystal structure of an ASBT homologue from Neisseria meningitidis ([ASBT.sub.NM]) in detergent was reported recently (9), showing the protein in an inward-open conformation bound to two [Na.sup.+] and a taurocholic acid. However, the structural changes that bring bile acid and [Na.sup.+] across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of [Na.sup.+] and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii ([ASBT.sub.Yf]) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved 'crossover' region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications for the location and orientation of the bile acid during transport, as well as for the translocation pathway for [Na.sup.+]., Purified [ASBT.sub.Yf], when reconstituted into liposomes, mediates [Na.sup.+]-dependent transport of the conjugated bile acid taurocholic acid (TCA) with an apparent Michaelis constant ([K.sub.m]) of 46.8 6 7.4 µM (Fig. 1a, [...]

TrkH belongs to a superfamily of [K.sup.+] transport proteins required for growth of bacteria in low external [K.sup.+] concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH resembles a [K.sup.+] channel and may have a gating mechanism substantially different from [K.sup.+] channels. TrkH assembles with TrkA, a cytosolic protein comprising two RCK (regulate the conductance of [K.sup.+]) domains, which are found in certain [K.sup.+] channels and control their gating. However, fundamental questions on whether TrkH is an ion channel and how it is regulated by TrkA remain unresolved. Here we show single-channel activity of TrkH that is upregulated by ATP via TrkA. We report two structures of the tetrameric TrkA ring, one in complex with TrkH and one in isolation, in which the ring assumes two markedly different conformations. These results suggest a mechanism for how ATP increases TrkH activity by inducing conformational changes in TrkA., [K.sup.+] is concentrated in all living cells and is essential for many physiological processes. In bacteria, homeostasis of [K.sup.+] is largely mediated by specialized [K.sup.+] transport proteins known as the [...]

Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His 250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation., Bacterial phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) [1] transport saccharides across the cell membrane and phosphorylate them before their release into the cytosol (2-7). Phosphorylation of the incoming saccharide primes it for [...]

The TrkH/TrkG/KtrB proteins mediate [K.sup.+] uptake in bacteria and probably evolved from simple [K.sup.+] channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of [K.sup.+] channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of [K.sup.+] and [Rb.sup.+] over smaller ions such as [Na.sup.+] or [Li.sup.+]. Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of [K.sup.+] flux. These results reveal the molecular basis of [K.sup.+] selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins., [K.sub.+] is highly concentrated in all living cells and plays diverse physiological roles such as setting membrane potential (1), regulating turgor pressure (2) and maintaining intracellular pH (3-5). Because [K.sup.+] [...]

Urea is highly concentrated in the mammalian kidney to produce the osmotic gradient necessary for water re-absorption. Free diffusion of urea across cell membranes is slow owing to its high polarity, and specialized urea transporters have evolved to achieve rapid and selective urea permeation. Here we present the 2.3 Å structure of a functional urea transporter from the bacterium Desulfovibrio vulgaris. The transporter is a homotrimer, and each subunit contains a continuous membrane-spanning pore formed by the two homologous halves of the protein. The pore contains a constricted selectivity filter that can accommodate several dehydrated urea molecules in single file. Backbone and side-chain oxygen atoms provide continuous coordination of urea as it progresses through the filter, and well-placed α-helix dipoles provide further compensation for dehydration energy. These results establish that the urea transporter operates by a channel-like mechanism and reveal the physical and chemical basis of urea selectivity., Urea is ubiquitous in nature. Bacteria take up urea and convert it to ammonia for use as a nitrogen source, and in certain enteric pathogens, a buffer for surviving the [...]

Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His 250 and Glu 334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation. [ABSTRACT FROM AUTHOR]

The Kvl family voltage-dependent K+ channels assemble with cytosolic β subunits (Kvβ). which are composed of a flexible N terminus followed by a structured core domain. The N terminus of certain Kvβs inactivates the channel by blocking the ion conduction pore, and the core domain is a functional enzyme that uses NADPH as a cofactor. Oxidation of the Kvβ-bound NADPH inhibits inactivation and potentiates channel current, but the mechanism behind this effect is unknown. Here we show that after oxidation, the core domain binds to part of the N terminus. thus restraining it from blocking the channel. The interaction is partially mediated by two negatively charged residues on the core domain and three positively charged ones on the N terminus. These results provide a molecular basis for the coupling between the cellular redox state and channel activity, and establish Kvβ as a target for pharmacological control of Kvl channels. [ABSTRACT FROM AUTHOR]

The TrkH/TrkG/KtrB proteins mediate K+ uptake in bacteria and probably evolved from simple K+ channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K+ channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K+ and Rb+ over smaller ions such as Na+ or Li+. Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K+ flux. These results reveal the molecular basis of K+ selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins. [ABSTRACT FROM AUTHOR]

Shared ancestry and introgression can contribute to genetic similarity between hybridizing species, and it is generally difficult to disentangle these causes. However, shared ancestry plays a more limited role in traits that have recently undergone parallel directional selection in the two species, permitting the role of introgression to be better understood. The butterflies Colias eurytheme (Boisduval) and Colias philodice (Godart) (Lepidoptera, Pieridae) are native to North America and have shifted their host ranges in parallel onto several introduced weedy and agricultural legumes. These butterflies hybridize at moderate rates throughout their range, and there is a strong possibility that they could be sharing host-associated adaptations. We split families of each species among nine introduced, prospective hosts and measured survivorship, larval duration, pupal weight, and a new variable, effective daily growth rate (DGR), analogous to a compound daily interest rate in economics. We found strong effects of host, sex, and family (species), but negligible effects of the host*species interaction that would indicate species-specific differences in performance on different hosts. We found species-specific life-history differences: C. eurytheme matured significantly later and reached a significantly larger body size than C. philodice while growing at the same DGR. Protandry was strong, and males, in addition to pupating sooner than females, grew significantly faster than females as measured by DGR. We measured broad-sense heritabilities and genetic correlations for host-associated performance variables. Most pairwise comparisons of performance among hosts and most pairwise comparisons between performance variables showed positive genetic correlations, except survivorship where little heritability was found. Nevertheless, a factorial multivariate analysis of variance of G-matrices showed highly significant species, host, and host*species interactions, suggesting differentially evolving genetic architectures underlying host adaptation in these two species, despite the small differences in overall performance. At least some of the genes affecting host performance in Colias are likely to be in the small, species-diagnostic regions and not shared via introgression between these hybridizing species. For biologists interested in the evolutionary ecology of their host associations, including applied biologists managing their agricultural pest potential, C. eurytheme and C. philodice are most usefully studied as if they were a single polymorphic species wherever they co-exist. In studying species that hybridize readily with a sympatric congener, it may often be necessary to include the second species in the experimental design. [ABSTRACT FROM AUTHOR]