Ferroportin (Fpn), i.e., solute carrier family 40 (SLC40A1), is the only known Fe2+ exporter in mammals. It mediates Fe2+ release from cells and is essential for maintaining the iron homeostasis. Fpn is also the only known target by an endogenous peptide hormone, hepcidin. We have solved several high resolution structures of Fpn bound with hepcidin and mini-hepcidin PR73, which clearly revealed the inhibition mechanism of these molecules. Fpn is embedded in the lipid bilayer of cellular membranes.

Selective permeation of K+ across membranes regulates the membrane potential, and all living organisms, aside from some parasites, carry at least one gene for a K+ channel. Several prokaryotic K+ channels have been implicated in various aspects of bacterial physiology, including electrical signaling and information propagation, but the vast majority remain unexplored regarding their mechanisms of activation and functions in cells. The KCH gene encodes for the only K+ channel in the E. coli genome.

Ferroportin (Fpn) is the only known iron exporter in humans and is essential for maintaining iron homeostasis. Fpn activity is suppressed by hepcidin, an endogenous peptide hormone, which inhibits iron export and promotes endocytosis of Fpn. Hepcidin deficiency leads to hemochromatosis and iron-loading anemia. Previous studies have shown that small peptides that mimic the first few residues of hepcidin, i.e., minihepcidins, are more potent than hepcidin. However, the mechanism of enhanced inhibition by minihepcidins remains unclear. Here, we report the structure of human ferroportin in complex with a minihepcidin, PR73 that mimics the first 9 residues of hepcidin, at 2.7 Å overall resolution. The structure reveals novel interactions that were not present between Fpn and hepcidin. We validate PR73-Fpn interactions through binding and transport assays. These results provide insights into how minihepcidins increase inhibition potency and will guide future development of Fpn inhibitors.

Ferroportin (Fpn) is a transporter that releases ferrous ion (Fe2+) from cells and is important for homeostasis of iron in circulation. Export of one Fe2+ by Fpn is coupled to import of two H+ to maintain charge balance. Here, we show that human Fpn (HsFpn) binds to and mediates Ca2+ transport. We determine the structure of Ca2+-bound HsFpn and identify a single Ca2+ binding site distinct from the Fe2+ binding sites. Further studies validate the Ca2+ binding site and show that Ca2+ transport is not coupled to transport of another ion. In addition, Ca2+ transport is significantly inhibited in the presence of Fe2+ but not vice versa. Function of Fpn as a Ca2+ uniporter may allow regulation of iron homeostasis by Ca2+.

Human sodium-dependent multivitamin transporter (hSMVT) recognizes and transports a variety of vitamins such as biotin, lipoate, pantothenate, and I-, and the concentrative uptake of the vitamin substrates is driven by the Na+ gradient. Since humans cannot synthesize pantothenate or biotin, their intestinal absorption by hSMVT is essential for normal health and development. hSMVT has also been employed for the targeted delivery of prodrugs (e.g., biotinylated acyclovir) to improve their bioavailability and in the “triple vitamin” (pantothenate, biotin and lipoate) replacement therapy to treat a neurodegenerative disorder. hSMVT is a member of the SLC5 gene family which includes a number of sodium solute symporters (SSS) such as sodium-dependent glucose transporters (SGLTs) and sodium-iodide symporter (NIS). Structures of bacterial homologs of SGLT have been reported and these structures have facilitated extensive structure-based mechanistic studies aimed at understanding the mechanism of Na+-dependent sugar binding and transport in human SGLT. Relatively little is known in terms substrate recognition and mechanism of transport in hSMVT and NIS. We expressed and purified crSMVT, a hSMVT homolog from Chlamydomonas reinhardtii (single cell green algae). Functional studies show that crSMVT binds to and transports pantothenate, bioton, and lipoic acid in Na+ dependent manner. We then solved the structure of crSMVT in lipidic cubic phase to 2.4 Å resolution by x-ray crystallography. The crSMVT structure identifies three Na+ binding sites and one substrate binding site. Since the bacterial homolog of SGLT has one Na+ binding site, we will validate the Na+ binding sites in crSMVT and determine their roles in substrate binding and transport. We will also determine how different substrates are accommodated by the substrate binding sites.

Copper uptake in eukaryotic cells is facilitated by the CTR copper transport proteins. Crystal structure of a CTR homolog from xx S. salar shows that CTR is a homotrimer and that each protomer has 3 transmembrane helices. A central permeation pathway is formed by the three subunits and two Cu binding sites were identified, each formed by three highly conserved methionines. It remains unknown how each of the Cu binding site contribute to selective permeation of Cu, and whether Cu transport is coupled to transport of another ion. We expressed and purified CTRs from human and C. elegans, and examined their functions in cell-free transport and binding assays. Because Cu+ is not stable in solution, we measured Ag+ transport in CTR. CTR transports Ag+ at a rate at least 10 fold higher than divalent transition metal ions such as nickel (Ni2+) or cobalt (Co2+). Mutations to the conserved methionines significantly reduce Ag+ transport but have little effect on Ni2+ transport. Ni2+ transport was instead affected by mutation to a conserved glutamate residue. These results indicate that CTR favors monovalent transition metal ions and that mono- and divalent cations may interact with CTR at different sites. This conclusion is also supported by affinity measurement of different ions. We also discovered that the ion transport process is electroneutral, indicating that permeation of Ag+ or other transition metal ions is accompanied by transport of another ion. After testing all possible candidates, we arrived at the conclusion that hydroxyl ions (OH−) are co-transported with the metal ions. It is not clear if CTR transports the two ions individually or together as a neutral metal hydroxide complex ion. Our study revealed for the first time the mechanism of ion selectivity and transport in CTR.

Ferroportin is the only cellular iron exporter in human and essential for iron homoeostasis. Mutations in ferroportin are associated with hemochromatosis or ferroportin diseases characterized by a paradoxical combination of anemia and abnormal accumulation of iron in cells. Ferroportin is also the target of hepcidin, which is a hormone that downregulates ferroportin activity. However, due to a lack of three-dimensional structures, the mechanism of iron transport by ferroportin and its regulation by hepcidin remains unclear. Here we present the structure of a ferroportin from the primate Philippine tarsier (TsFpn) at 3.0 Å resolution determined by cryo-electron microscopy. TsFpn has a structural fold common to major facilitator superfamily of transporters and the current structure is in an outward-open conformation. The structure identifies two potential ion binding sites with each site coordinated by two residues. Functional studies demonstrate that TsFpn is a H + /Fe 2+ antiporter and that transport of one Fe 2+ is coupled to the transport of two H + in the opposite direction such that the transport cycle is electroneutral. Further studies show that the two ion binding sites affect transport of H + and Fe 2+ differently. The structure also provides mechanistic interpretation for mutations that cause ferroportin diseases.

Ferroportin is an iron exporter essential for releasing cellular iron into circulation. Ferroportin is inhibited by a peptide hormone, hepcidin. In humans, mutations in ferroportin lead to ferroportin diseases that are often associated with accumulation of iron in macrophages and symptoms of iron deficiency anemia. Here we present the structures of the ferroportin from the primate Philippine tarsier (TsFpn) in the presence and absence of hepcidin solved by cryo-electron microscopy. TsFpn is composed of two domains resembling a clamshell and the structure defines two metal ion binding sites, one in each domain. Both structures are in an outward-facing conformation, and hepcidin binds between the two domains and reaches one of the ion binding sites. Functional studies show that TsFpn is an electroneutral H + /Fe 2+ antiporter so that transport of each Fe 2+ is coupled to transport of two H + in the opposite direction. Perturbing either of the ion binding sites compromises the coupled transport of H + and Fe 2+ . These results establish the structural basis of metal ion binding, transport and inhibition in ferroportin and provide a blueprint for targeting ferroportin in pharmacological intervention of ferroportin diseases.

Cyclic nucleotide–gated (CNG) channels convert cyclic nucleotide (CN) binding and unbinding into electrical signals in sensory receptors and neurons. The molecular conformational changes underpinning ligand activation are largely undefined. We report both closed- and open-state atomic cryo-EM structures of a full-length Caenorhabditis elegans cyclic GMP−activated channel TAX-4, reconstituted in lipid nanodiscs. These structures, together with computational and functional analyses and a mutant channel structure, reveal a double-barrier hydrophobic gate formed by two S6 amino acids in the central cavity. cGMP binding produces global conformational changes that open the cavity gate located ~52 Å away but do not alter the structure of the selectivity filter—the commonly presumed activation gate. Our work provides mechanistic insights into the allosteric gating and regulation of CN-gated and nucleotide-modulated channels and CNG channel−related channelopathies. Cryo-EM structures of a C. elegans cGMP-activated channel TAX-4 in lipid nanodiscs reveal a hydrophobic gate in the central cavity and, together with electrophysiology, provide mechanistic insights into the gating and regulation of CNG channels.

TrkH is a bacterial ion channel implicated in K+ uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.

Copper (Cu) is a cofactor in a number of oxidoreductases and is an essential nutrient for human. The CTR copper transporters are the major protein for selective Cu uptake. Previous functional studies of CTR have relied on cell-based assays, and are limited by complications caused by uncertainty in the amount of surface expressed CTR protein and by potential interference from other endogenous transporters and channels. We have expressed and purified CTR proteins of two eukaryotic species, and measured ion binding and transport. Using isothermal titration calorimetry, we found that CTRs have 10 fold higher affinity for the monovalent transition metal ion Ag+ than the divalent transition metal ions such as Co2+, Ni2+.and Zn2+. Using a liposome-based flux assay, we found that CTRs transport Ag+ at least 10 fold faster than the divalent cations. These results are consistent with previous observations and provide quantification for ion binding and transport. We then examined the role of the two highly conserved methionine residues and found opposing effects on ion binding and transport. These results provided hints of a novel mechanism of ion selectivity and transport, and inspired further experiments directly test the mechanism.

The superfamily of K+ transporters (SKT) is ubiquitous in bacteria, fungi and plants. SKT proteins are required for survival of bacteria in low K+ conditions and are involved in salt regulation in fungi and plants. Bacterial SKTs have two components, a membrane embedded protein TrkH that forms a homodimer with each protomer resembling an ion channel, and a cytosolic protein TrkA that forms a homotetramer with each protomer containing two RCK domains. Single-channel activities of the TrkH-TrkA complex were recorded and analyzed: ATP or ATP analogs such as AMPPNP and ATPγS activate the channel while ADP closes it. In order to understand how ADP and ATP regulate the ion channel, we solved the structures of TrkH-TrkA in the presence of ADP, AMPPNP, ATPγS or ATP by either x-ray crystallography or cryo-electron microscopy in atomic resolution. These structures show different conformations of TrkH and different shapes of the TrkA tetramer, and suggest a novel mechanism of gating. We then applied mutational analyses to validate key components of the gating mechanism.

The superfamily of K+ transporters (SKT) is ubiquitous in bacteria, fungi and plants. SKT proteins are required for survival of bacteria in low K+ conditions and are involved in salt regulation in fungi and plants. Bacterial SKTs have two components, a membrane embedded protein that resembles an ion channel and a cytosolic protein that regulates channel gating [1]. Crystal structures of two bacterial SKT systems were reported recently [2,3]. In both structures, the membrane embedded component forms a homodimer onto which a homotetrameric ring of the cytosolic protein docks. Single-channel activities of one of the complexes, the TrkH (membrane embedded) -TrkA (cytosolic) complex, were recorded and analyzed: ATP or ATP analogs such as AMPPNP activates the channel while ADP closes it [2]. The structure of the TrkH-TrkA complex is likely in a closed conformation because it was crystallized in the presence of NADH, a ligand that does not activates the channel. In order to understand how ATP or its analogs induces channel opening, we solved the structure of the TrkH-TrkA complex in the presence of AMPPNP to 3.29 Å by x-ray crystallography. When compared to the previous structures, the new structure shows that each TrkA protomer binds to two AMPPNP molecules, and that the TrkA tetramer assumes an elongated conformation that likely induces a change in the TrkH. Conformational changes in the TrkH involve significant changes in the dimer interface and are different from any other channels of known structure. These new observations and hypotheses will be validated and tested by mutational and functional analyses. [1] Levin EJ, Zhou M. Recent progress on the structure and function of the TrkH/KtrB ion channel. Curr Opin Struct Biol. 2014;27:95-101. [2] Cao Y, Pan Y, Huang H, et al. Gating of the TrkH ion channel by its associated RCK protein TrkA. Nature. 2013;496(7445):317-22. [3] Vieira-pires RS, Szollosi A, Morais-cabral JH. The structure of the KtrAB potassium transporter. Nature. 2013;496(7445):323-8.

Nucleotide-binding leucine-rich repeat (NLR) proteins serve as immune receptors in both plants and animals. To identify components required for NLR-mediated immunity, we designed and carried out a chemical genetics screen to search for small molecules that can alter immune responses in Arabidopsis thaliana. From 13 600 compounds, we identified Ro 8-4304 that was able to specifically suppress the severe autoimmune phenotypes of chs3-2D (chilling sensitive 3, 2D), including the arrested growth morphology and heightened PR (Pathogenesis Related) gene expression. Further, six Ro 8-4304 insensitive mutants were uncovered from the Ro 8-4304-insensitive mutant (rim) screen using a mutagenized chs3-2D population. Positional cloning revealed that rim1 encodes an allele of AtICln (I, currents; Cl, chloride; n, nucleotide). Genetic and biochemical analysis demonstrated that AtICln is in the same protein complex with the methylosome components small nuclear ribonucleoprotein D3b (SmD3b) and protein arginine methyltransferase 5 (PRMT5), which are required for the biogenesis of small nuclear ribonucleoproteins (snRNPs) involved in mRNA splicing. Double mutant analysis revealed that SmD3b is also involved in the sensitivity to Ro 8-4304, and the prmt5-1 chs3-2D double mutant is lethal. Loss of AtICln, SmD3b, or PRMT5 function results in enhanced disease resistance against the virulent oomycete pathogen Hyaloperonospora arabidopsidis Noco2, suggesting that mRNA splicing plays a previously unknown negative role in plant immunity. The successful implementation of a high-throughput chemical genetic screen and the identification of a small-molecule compound affecting plant immunity indicate that chemical genetics is a powerful tool to study whole-organism plant defense pathways.

A high intracellular potassium ion concentration is required for many essential cellular functions. To carry potassium ions across membranes, organisms must express potassium ion transport proteins, such as proteins in the Superfamily of Potassium Transporters (SKT). TrkH, a member of SKT, is required for bacterial growth in environments with low external potassium concentration. Previous studies showed that TrkH is an ion channel and ATP increases channel activity through an associated cytosolic protein, TrkA, which forms a homotetrameric ring. However, whether ATP regulation is preserved in TrkH of other organisms, and how ATP upregulates TrkH via TrkA are still not clear. Crystal structures of TrkH and TrkA suggest that movement of a tilted helix in TrkH and a conformational change in the TrkA tetrameric ring are required for the gating process. We have expressed and purified TrkH and TrkA from various pathogens, reconstituted them into liposomes and will examine the effects of ATP and other potential ligands on their activity. We will test our structure-inspired gating model by measuring the rate of crosslinking between strategically placed pairs of cysteine mutations.

Inhibitors of the bile acid transporter ASBT may be useful therapeutics for treating hypercholesterolaemia and type 2 diabetes; here, two X-ray crystal structures of an ASBT homologue from Yersinia frederiksenii are solved. This paper reports two X-ray crystal structures of a bacterial homologue of the human apical sodium-dependent bile salt transporter (ASBT, also known as SLC10A2), one of two transporters involved in retrieving secreted bile acids from the intestine. The homologue (termed ASBTYf), from Yersinia frederiksenii, was crystallized in a lipid environment. The structures reveal that a large rigid-body rotation of a substrate-binding domain gives alternate accessibility to the highly conserved 'crossover' region, where two discontinuous transmembrane helices cross each other. This result has implications for the location and orientation of the bile acid during transport, as well as for the translocation pathway for sodium ions. The authors cite evidence that implies that overall fold and transport mechanism are similar between ASBT and ASBTYf and they suggest that ASBTYf may serve as a useful model system for understanding mechanisms of transport and inhibition in the mammalian ASBT homologues. ASBT inhibitors are being studied as potential therapeutics for the treatment of hypercholesterolaemia and type II diabetes. 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 resecretion1. In humans, there are two Na+-dependent bile acid transporters involved in enterohepatic recirculation, the Na+-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 ileum2. 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 consumption3,4. 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 data2,5,6,7,8. The crystal structure of an ASBT homologue from Neisseria meningitidis (ASBTNM) in detergent was reported recently9, showing the protein in an inward-open conformation bound to two Na+ and a taurocholic acid. However, the structural changes that bring bile acid and Na+ across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of Na+ and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii (ASBTYf) 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+.

TrkH and its homologs TrkG and KtrB belong to a superfamily of K+ transport proteins that are required for growth of bacteria in low external K+ concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH forms a homodimer, and each protomer resembles a K+ channel with a unique gating mechanism. TrkH assembles with TrkA, a soluble protein comprising two Regulate-Conductance-of-K+, or RCK domains, which control the gating of certain K+ channels. In K+ channels, eight RCK domains form a four-fold symmetric gating ring that matches the four-fold symmetry of the channel. A dilation of the diameter of the gating ring directly translates into expansion of the pore-lining helices of the channel and hence opening of the permeation pathway. However, the gating ring expansion mechanism in K+ channels appears incompatible with the dimeric architecture and the different gating mechanism in TrkH. In addition, although TrkH resembles a K+ channel, its channel activity has never been demonstrated. We have recorded single-channel activity from TrkH in spheroplasts consistent with two partly coupled pores. We also found that channel activity is upregulated by ATP via TrkA. To understand how channel gating is regulated, we solved two structures of the TrkA tetrameric ring, one in complex with TrkH and one in isolation, in which the ring assumes two dramatically different conformations. The structures suggest a mechanism for how ATP increases the open probability of the TrkH ion channel by inducing conformational changes in TrkA.

TrkH belongs to a superfamily of K(+) transport proteins required for growth of bacteria in low external K(+) concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH resembles a K(+) channel and may have a gating mechanism substantially different from K(+) channels. TrkH assembles with TrkA, a cytosolic protein comprising two RCK (regulate the conductance of K(+)) domains, which are found in certain K(+) 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.