Structural basis and synergism of ATP and Na+ activation in bacterial K+ uptake system KtrAB.

The K+ uptake system KtrAB is essential for bacterial survival in low K+ environments. The activity of KtrAB is regulated by nucleotides and Na+. Previous studies proposed a putative gating mechanism of KtrB regulated by KtrA upon binding to ATP or ADP. However, how Na+ activates KtrAB and the Na+ binding site remain unknown. Here we present the cryo-EM structures of ATP- and ADP-bound KtrAB from Bacillus subtilis (BsKtrAB) both solved at 2.8 Å. A cryo-EM density at the intra-dimer interface of ATP-KtrA was identified as Na+, as supported by X-ray crystallography and ICP-MS. Thermostability assays and functional studies demonstrated that Na+ binding stabilizes the ATP-bound BsKtrAB complex and enhances its K+ flux activity. Comparing ATP- and ADP-BsKtrAB structures suggests that BsKtrB Arg417 and Phe91 serve as a channel gate. The synergism of ATP and Na+ in activating BsKtrAB is likely applicable to Na+-activated K+ channels in central nervous system.

Dimerization of the CNNM extracellular domain.

Cystathionine- ß $$ \beta $$ -synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) are an evolutionarily conserved family of magnesium transporters. They mediate magnesium homeostasis directly by transport of Mg2+ ions and indirectly by regulation of the transient receptor potential ion channel subfamily M member 7 (TRPM7). Here, we report the crystal structure of the extracellular domain of tapeworm CNNM4. The domain forms a dimer of immunoglobulin-like (Ig-like) folds with electron density observed for three glycosylation sites. Analytical ultracentrifugation confirms that mutations in the extracellular domain of human CNNM4 prevent its dimerization. An analogous mutation in mouse CNNM2 impairs its activity in a cellular assay of Mg2+ transport.

AMPK phosphorylates and stabilises copper transporter 1 to synergise metformin and copper chelator for breast cancer therapy.

BACKGROUND: Predominant roles of copper and its transporter, copper transporter 1 (CTR1), in tumorigenesis have been explored recently; however, the upstream regulation of CTR1 and combinational intervention of copper chelators in malignancies remain largely unclear. METHODS: CRISPR/Cas9-based kinome screening was used to identify the CTR1 upstream kinases. Immunofluorescence assays were utilised to detect CTR1 localisation. In vitro kinase assays and mass spectrometry were performed to detect CTR1 phosphorylation. Ubiquitination assays were performed to validate CTR1 stability. Colony formation, EdU labelling, Annexin V-FITC/PI-based apoptosis assays were carried out to detect the drug effect on cell growth and apoptosis. Xenografted mouse models were employed to investigate drug effects in vivo. RESULTS: We identify that CTR1 undergoes AMPK-mediated phosphorylation, which enhances CTR1 stabilisation and membrane translocation by affecting Nedd4l interaction, resulting in increased oncogenic roles in breast cancer. Importantly, activation of AMPK with its agonist metformin markedly enhances CTR1 levels, and leads to the combinational usage of AMPK agonists and copper chelators for breast cancer treatment. CONCLUSIONS: Our findings not only reveal the crosstalk between energy response and copper uptake via AMPK-mediated CTR1 phosphorylation and stability but also highlight the strategy to combat breast cancer by a combination of AMPK agonists and copper chelators. SIGNIFICANCE: The connection between energy response and copper homoeostasis is linked by AMPK phosphorylating and stabilising CTR1, which provides a promising strategy to combat breast cancer by combining AMPK agonists and copper chelators.

Copper coordination states affect the flexibility of copper Metallochaperone Atox1: Insights from molecular dynamics simulations.

Copper is an essential element in nature but in excess, it is toxic to the living cell. The human metallochaperone Atox1 participates in copper homeostasis and is responsible for copper transmission. In a previous multiscale simulation study, we noticed a change in the coordination state of the Cu(I) ion, from 4 bound cysteine residues to 3, in agreement with earlier studies. Here, we perform and analyze classical molecular dynamic simulations of various coordination states: 2, 3, and 4. The main observation is an increase in protein flexibility as a result of a decrease in the coordination state. In addition, we identified several populated conformations that correlate well with double electron-electron resonance distance distributions or an X-ray structure of Cu(I)-bound Atox1. We suggest that the increased flexibility might benefit the process of ion transmission between interacting proteins. Further experiments can scrutinize this hypothesis and shed additional light on the mechanism of action of Atox1.

Inhibited KdpFABC transitions into an E1 off-cycle state.

KdpFABC is a high-affinity prokaryotic K+ uptake system that forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB). At high K+ levels, KdpFABC needs to be inhibited to prevent excessive K+ accumulation to the point of toxicity. This is achieved by a phosphorylation of the serine residue in the TGES162 motif in the A domain of the pump subunit KdpB (KdpBS162-P). Here, we explore the structural basis of inhibition by KdpBS162 phosphorylation by determining the conformational landscape of KdpFABC under inhibiting and non-inhibiting conditions. Under turnover conditions, we identified a new inhibited KdpFABC state that we termed E1P tight, which is not part of the canonical Post-Albers transport cycle of P-type ATPases. It likely represents the biochemically described stalled E1P state adopted by KdpFABC upon KdpBS162 phosphorylation. The E1P tight state exhibits a compact fold of the three cytoplasmic domains and is likely adopted when the transition from high-energy E1P states to E2P states is unsuccessful. This study represents a structural characterization of a biologically relevant off-cycle state in the P-type ATPase family and supports the emerging discussion of P-type ATPase regulation by such states.

Polarized trafficking and copper transport activity of ATP7B: A mutational approach to establish genotype-phenotype correlation in Wilson disease.

Mutation in ATP7B gene causes Wilson disease (WD) that is characterized by severe hepatic and neurological symptoms. ATP7B localizes at the trans-Golgi Network (TGN) transporting copper to copper-dependent enzymes and traffics in apically targeted vesicles upon intracellular copper elevation. To decode the cellular underpinnings of WD manifestation we investigated copper-responsive polarized trafficking and copper transport activity of 15 WD causing point mutations in ATP7B. Amino-terminal mutations Gly85Val, Leu168Pro, and Gly591Asp displayed TGN and subapical localization whereas, Leu492Ser mislocalized at the basolateral region. The actuator domain mutation Gly875Arg shows retention in the endoplasmic reticulum (ER), Ala874Val and Leu795Phe show partial targeting to TGN and post-Golgi vesicles. The nucleotide-binding domain mutations His1069Gln and Leu1083Phe also display impaired targeting. The C-terminal mutations Leu1373Pro/Arg is arrested at ER but Ser1423Asn shows TGN localization. Transmembrane mutant Arg778Leu resides in ER and TGN while Arg969Gln is exclusively ER localized. Cellular Cu level does not alter the targeting of any of the studied mutations. Mutants that traffic to TGN exhibits biosynthetic function. Finally, we correlated cellular phenotypes with the clinical manifestation of the two most prevalent mutations; the early onset and more aggressive WD caused by Arg778Leu and the milder form of WD caused by mutation His1069Gln.

Crystal structures of EfeB and EfeO in a bacterial siderophore-independent iron transport system.

EfeUOB is a siderophore-independent iron uptake mechanism in bacteria. EfeU, EfeO, and EfeB are a permease, an iron-binding or electron-transfer protein, and a peroxidase, respectively. A Gram-negative bacterium, Sphingomonas sp. strain A1, encodes EfeU, EfeO, EfeB together with alginate-binding protein Algp7, a truncated EfeO-like protein (EfeOII), in the genome. The typical EfeO (EfeOI) consists of N-terminal cupredoxin and C-terminal M75 peptidase domains. Here, we detail the structure and function of bacterial EfeB and EfeO. Crystal structures of strain A1 EfeB and Escherichia coli EfeOI were determined at 2.30 Å and 1.85 Å resolutions, respectively. A molecule of heme involved in oxidase activity was bound to the C-terminal Dyp peroxidase domain of EfeB. Two domains of EfeOI were connected by a short loop, and a zinc ion was bound to four residues, Glu156, Glu159, Asp173, and Glu255, in the C-terminal M75 peptidase domain. These residues formed tetrahedron geometry suitable for metal binding and are well conserved among various EfeO proteins including Algp7 (EfeOII), although the metal-binding site (HxxE) is proposed in the C-terminal M75 peptidase domain. This is the first report on structure of a typical EfeO with two domains, postulating a novel metal-binding motif "ExxE-//-D-//-E" in the EfeO C-terminal M75 peptidase domain.

A review on bacterial redox dependent iron transporters and their evolutionary relationship.

Iron is an essential yet toxic micronutrient and its transport across biological membranes is tightly regulated in all living organisms. One such iron transporter, the Ftr-type permeases, is found in both eukaryotic and prokaryotic cells. These Ftr-type transporters are required for iron transport, predicted to form α-helical transmembrane structures, and conserve two ArgGluxxGlu (x = any amino acid) motifs. In the yeast Ftr transporter (Ftr1p), a ferroxidase (Fet3p) is required for iron transport in an oxidation coupled transport step. None of the bacterial Ftr-type transporters (EfeU and FetM from E. coli; cFtr from Campylobacter jejuni; FtrC from Brucella, Bordetella, and Burkholderia spp.) contain a ferroxidase protein. Bioinformatics report predicted periplasmic EfeO and FtrB (from the EfeUOB and FtrABCD systems) as novel cupredoxins. The Cu2+ binding and the ferrous oxidation properties of these proteins are uncharacterized and the other two bacterial Ftr-systems are expressed without any ferroxidase/cupredoxin, leading to controversy about the mode of function of these transporters. Here, we review published data on Ftr-type transporters to gain insight into their functional diversity. Based on original bioinformatics data presented here evolutionary relations between these systems are presented.

The Zinc and Iron Binuclear Transport Center of ZupT, a ZIP Transporter from Escherichia coli.

ZupT fromEscherichia coliis a member of the Zrt-/Irt-like Protein (ZIP) transporter family, which is responsible for zinc uptake during zinc-sufficient conditions. ZIP transporters have been shown to transport different divalent metal ions including zinc, iron, manganese, and cadmium. In this study, we show that ZupT has an asymmetric binuclear metal center in the transmembrane domain; one metal-binding site, M1, binds zinc, cadmium, and iron, while the other, M2, binds iron only and with higher affinity than M1. Using site-specific mutagenesis and transport activity measurements in whole cells and proteoliposomes, we show that zinc is transported from M1, while iron is transported from M2. The two sites share a common bridging ligand, a conserved glutamate residue. M1 and M2 have ligands from highly conserved motifs in transmembrane domains 4 and 5. Additionally, M2 has a ligand from transmembrane domain 6, a glutamate residue, which is conserved in the gufA subfamily of ZIP transporters, including ZupT and the human ZIP11. Unlike cadmium, iron transport from M2 does not inhibit the zinc transport activity but slightly stimulates it. This stimulation of activity is mediated through the bridging carboxylate ligand. The binuclear zinc-iron binding center in ZupT has likely evolved to enable the transport of essential metals from two different sites without competition; a similar mechanism of metal transport is likely to be found in the gufA subfamily of ZIP transporter proteins.

Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC.

KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-π stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.

Genome-Wide Identification of the Nramp Gene Family in Spirodela polyrhiza and Expression Analysis under Cadmium Stress.

Natural resistance-associated macrophage proteins (Nramps) are specific metal transporters in plants with different functions among various species. The evolutionary and functional information of the Nramp gene family in Spirodela polyrhiza has not been previously reported in detail. To identify the Nramp genes in S. polyrhiza, we performed genome-wide identification, characterization, classification, and cis-elements analysis among 22 species with 138 amino acid sequences. We also conducted chromosomal localization and analyzed the synteny relationship, promoter, subcellular localization, and expression patterns in S. polyrhiza. ß-Glucuronidase staining indicated that SpNramp1 and SpNramp3 mainly accumulated in the root and joint between mother and daughter frond. Moreover, SpNramp1 was also widely displayed in the frond. SpNramp2 was intensively distributed in the root and frond. Quantitative real-time PCR results proved that the SpNramp gene expression level was influenced by Cd stress, especially in response to Fe or Mn deficiency. The study provides detailed information on the SpNramp gene family and their distribution and expression, laying a beneficial foundation for functional research.

Hepcidin-Ferroportin Interaction Controls Systemic Iron Homeostasis.

Despite its abundance in the environment, iron is poorly bioavailable and subject to strict conservation and internal recycling by most organisms. In vertebrates, the stability of iron concentration in plasma and extracellular fluid, and the total body iron content are maintained by the interaction of the iron-regulatory peptide hormone hepcidin with its receptor and cellular iron exporter ferroportin (SLC40a1). Ferroportin exports iron from duodenal enterocytes that absorb dietary iron, from iron-recycling macrophages in the spleen and the liver, and from iron-storing hepatocytes. Hepcidin blocks iron export through ferroportin, causing hypoferremia. During iron deficiency or after hemorrhage, hepcidin decreases to allow iron delivery to plasma through ferroportin, thus promoting compensatory erythropoiesis. As a host defense mediator, hepcidin increases in response to infection and inflammation, blocking iron delivery through ferroportin to blood plasma, thus limiting iron availability to invading microbes. Genetic diseases that decrease hepcidin synthesis or disrupt hepcidin binding to ferroportin cause the iron overload disorder hereditary hemochromatosis. The opposite phenotype, iron restriction or iron deficiency, can result from genetic or inflammatory overproduction of hepcidin.

Structural basis for potassium transport in prokaryotes by KdpFABC.

KdpFABC is an oligomeric K+ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K+ transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryogenic electron microscopy. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC, and KdpF remain static. We observe a series of spherical densities that we assign as K+ or water and which define a pathway for K+ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K+ transfer between alternative sites in KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K+ to the cytoplasm.

Metabolic iron detection through divalent metal transporter 1 and ferroportin mediated cocktail fluorogenic probes.

A cocktail [1 + 2] dual-fluorescent probe system was developed to realize the real-time visualization of dynamic iron state changes between Fe2+ and Fe3+ at the cellular level and in multicellular organisms, providing insights into the effect of DMT1 and ferroportin on iron regulation.

Overproduction of Human Zip (SLC39) Zinc Transporters in Saccharomyces cerevisiae for Biophysical Characterization.

Zinc constitutes the second most abundant transition metal in the human body, and it is implicated in numerous cellular processes, including cell division, DNA and protein synthesis as well as for the catalytic activity of many enzymes. Two major membrane protein families facilitate zinc homeostasis in the animal kingdom, i.e., Zrt/Irt-like proteins (ZIPs aka solute carrier 39, SLC39, family) and Zn transporters (ZnTs), essentially conducting zinc flux in the opposite directions. Human ZIPs (hZIPs) regulate import of extracellular zinc to the cytosol, being critical in preventing overaccumulation of this potentially toxic metal, and crucial for diverse physiological and pathological processes, including development of neurodegenerative disorders and several cancers. To date, our understanding of structure-function relationships governing hZIP-mediated zinc transport mechanism is scarce, mainly due to the notorious difficulty in overproduction of these proteins for biophysical characterization. Here we describe employment of a Saccharomyces cerevisiae-based platform for heterologous expression of hZIPs. We demonstrate that yeast is able to produce four full-length hZIP members belonging to three different subfamilies. One target (hZIP1) is purified in the high quantity and homogeneity required for the downstream biochemical analysis. Our work demonstrates the potential of the described production system for future structural and functional studies of hZIP transporters.

Novel variant in the CNNM2 gene associated with dominant hypomagnesemia.

The maintenance of magnesium (Mg2+) homeostasis is essential for human life. The Cystathionine-ß-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) have been described to be involved in maintaining Mg2+ homeostasis. Among these CNNMs, CNNM2 is expressed in the basolateral membrane of the kidney tubules where it is involved in Mg2+ reabsorption. A total of four patients, two of them with a suspected disorder of calcium metabolism, and two patients with a clinical diagnosis of primary tubulopathy were screened for mutations by Next-Generation Sequencing (NGS). We found one novel likely pathogenic variant in the heterozygous state (c.2384C>A; p.(Ser795*)) in the CNNM2 gene in a family with a suspected disorder of calcium metabolism. In this family, hypomagnesemia was indirectly discovered. Moreover, we observed three novel variants of uncertain significance in heterozygous state in the other three patients (c.557G>C; p.(Ser186Thr), c.778A>T; p.(Ile260Phe), and c.1003G>A; p.(Asp335Asn)). Our study shows the utility of Next-Generation Sequencing in unravelling the genetic origin of rare diseases. In clinical practice, serum Mg2+ should be determined in calcium and PTH-related disorders.

Dynamical Behavior of the Human Ferroportin Homologue from Bdellovibrio bacteriovorus: Insight into the Ligand Recognition Mechanism.

Members of the major facilitator superfamily of transporters (MFS) play an essential role in many physiological processes such as development, neurotransmission, and signaling. Aberrant functions of MFS proteins are associated with several diseases, including cancer, schizophrenia, epilepsy, amyotrophic lateral sclerosis and Alzheimer's disease. MFS transporters are also involved in multidrug resistance in bacteria and fungi. The structures of most MFS members, especially those of members with significant physiological relevance, are yet to be solved. The lack of structural and functional information impedes our detailed understanding, and thus the pharmacological targeting, of these transporters. To improve our knowledge on the mechanistic principles governing the function of MSF members, molecular dynamics (MD) simulations were performed on the inward-facing and outward-facing crystal structures of the human ferroportin homologue from the Gram-negative bacterium Bdellovibrio bacteriovorus (BdFpn). Several simulations with an excess of iron ions were also performed to explore the relationship between the protein's dynamics and the ligand recognition mechanism. The results reinforce the existence of the alternating-access mechanism already described for other MFS members. In addition, the reorganization of salt bridges, some of which are conserved in several MFS members, appears to be a key molecular event facilitating the conformational change of the transporter.

Serine phosphorylation regulates the P-type potassium pump KdpFABC.

KdpFABC is an ATP-dependent K+ pump that ensures bacterial survival in K+-deficient environments. Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down-regulation when K+ levels are restored has not been described. Here, we show that KdpFABC is inhibited when cells return to a K+-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the pump. Analyses of the transport cycle show that serine phosphorylation abolishes the K+-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type pumps and this study furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.

Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms.

The serum level of iron in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing the internalization and degradation of ferroportin1. Aberrant ferroportin activity can lead to diseases of iron overload, such as haemochromatosis, or iron limitation anaemias2. Here we determine cryogenic electron microscopy structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with hepcidin and the iron mimetic cobalt. These structures and accompanying molecular dynamics simulations identify two metal-binding sites within the N and C domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy terminus of hepcidin directly contacts the divalent metal in the ferroportin C domain. Hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, in which only ferroportin molecules loaded with iron are targeted for degradation. More broadly, our structural and functional insights may enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.