Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori.

Essential trace metals play key roles in the survival, replication, and virulence of bacterial pathogens. Helicobacter pylori (H. pylori), the main bacterial cause of gastric ulcers, requires Ni(ii) to colonize and persist in the acidic environment inside the stomach, exploiting the nickel-containing enzyme urease to catalyze the hydrolysis of urea to ammonia and bicarbonate and create a pH-buffered microenvironment. Urease utilizes Ni(ii) as a catalytic cofactor for its activity. In ureolytic bacteria, unique transmembrane (TM) transporters evolved to guarantee the selective uptake and efflux of Ni(ii) across cellular membranes to meet the cellular requirements. NixA is an essential Ni(ii) transporter expressed by H. pylori when the extracellular environment experiences a drop in pH. This Class I nickel-cobalt transporter of the NiCoT family catalyzes the uptake of Ni(ii) across the inner membrane from the periplasm. In this study, we characterized NixA using a platform whereby, for the first time on a NiCoT transporter, recombinantly expressed and purified NixA and key mutants in the translocation pathway have been reconstituted in artificial lipid bilayer vesicles (proteoliposomes). Fluorescent sensors responsive to Ni(ii) transport (Fluozin-3-Zn(ii)), luminal pH changes (pyranine), and membrane potential (oxonol VI) were encapsulated in the proteoliposomes lumen to monitor, in real-time, NixA transport properties and translocation mechanism. Kinetic transport analysis revealed that NixA is highly selective for Ni(ii) with no substrate promiscuity towards Co(ii), the other putative metal substrate of the NiCoT family, nor Zn(ii). NixA-mediated Ni(ii) transport exhibited a Michaelis-Menten-type saturable substrate concentration dependence, with an experimental KM, Ni(ii) = 31.0 ± 1.2 µM. Ni(ii) transport by NixA was demonstrated to be electrogenic, and metal translocation did not require a proton motive force, resulting in the generation of a positive-inside transmembrane potential in the proteoliposome lumen. Mutation analysis characterized key transmembrane residues for substrate recognition, binding, and/or transport, suggesting the presence of a three-step transmembrane translocation conduit. Taken together, these investigations reveal that NixA is a Ni(ii)-selective Class I NiCoT electrogenic uniporter. The work also provides an in vitro approach to characterize the transport properties of metal transporters responsible for Ni(ii) acquisition and extrusion in prokaryotes.

Plastic recognition and electrogenic uniport translocation of 1st-, 2nd-, and 3rd-row transition and post-transition metals by primary-active transmembrane P1B-2-type ATPase pumps.

Transmembrane P1B-type ATPase pumps catalyze the extrusion of transition metal ions across cellular lipid membranes to maintain essential cellular metal homeostasis and detoxify toxic metals. Zn(ii)-pumps of the P1B-2-type subclass, in addition to Zn2+, select diverse metals (Pb2+, Cd2+ and Hg2+) at their transmembrane binding site and feature promiscuous metal-dependent ATP hydrolysis in the presence of these metals. Yet, a comprehensive understanding of the transport of these metals, their relative translocation rates, and transport mechanism remain elusive. We developed a platform for the characterization of primary-active Zn(ii)-pumps in proteoliposomes to study metal selectivity, translocation events and transport mechanism in real-time, employing a "multi-probe" approach with fluorescent sensors responsive to diverse stimuli (metals, pH and membrane potential). Together with atomic-resolution investigation of cargo selection by X-ray absorption spectroscopy (XAS), we demonstrate that Zn(ii)-pumps are electrogenic uniporters that preserve the transport mechanism with 1st-, 2nd- and 3rd-row transition metal substrates. Promiscuous coordination plasticity, guarantees diverse, yet defined, cargo selectivity coupled to their translocation.

Rational Design of the ß-Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing.

Detection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the ß-bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn-off sensor SulfOFF-1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the ß-barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.

Discovery of a monomeric green fluorescent protein sensor for chloride by structure-guided bioinformatics.

Chloride is an essential anion for all forms of life. Beyond electrolyte balance, an increasing body of evidence points to new roles for chloride in normal physiology and disease. Over the last two decades, this understanding has been advanced by chloride-sensitive fluorescent proteins for imaging applications in living cells. To our surprise, these sensors have primarily been engineered from the green fluorescent protein (GFP) found in the jellyfish Aequorea victoria. However, the GFP family has a rich sequence space that could already encode for new sensors with desired properties, thereby minimizing protein engineering efforts and accelerating biological applications. To efficiently sample this space, we present and validate a stepwise bioinformatics strategy focused first on the chloride binding pocket and second on a monomeric oligomerization state. Using this, we identified GFPxm163 from GFPxm found in the jellyfish Aequorea macrodactyla. In vitro characterization shows that the binding of chloride as well as bromide, iodide, and nitrate rapidly tunes the ground state chromophore equilibrium from the phenolate to the phenol state generating a pH-dependent, turn-off fluorescence response. Furthermore, live-cell fluorescence microscopy reveals that GFPxm163 provides a reversible, yet indirect readout of chloride transport via iodide exchange. With this demonstration, we anticipate that the pairing of bioinformatics with protein engineering methods will provide an efficient methodology to discover and design new chloride-sensitive fluorescent proteins for cellular applications.

Comparative cisplatin reactivity towards human Zn7-metallothionein-2 and MTF-1 zinc fingers: potential implications in anticancer drug resistance.

Cis-diamminedichloroplatinum(II) (cisplatin) is a widely used metal-based chemotherapeutic drug for the treatment of cancers. However, intrinsic and acquired drug resistance limit the efficacy of cisplatin-based treatments. Increased production of intracellular thiol-rich molecules, in particular metallothioneins (MTs), which form stable coordination complexes with the electrophilic cisplatin, results in cisplatin sequestration leading to pre-target resistance. MT-1/-2 are overexpressed in cancer cells, and their expression is controlled by the metal response element (MRE)-binding transcription factor-1 (MTF-1), featuring six Cys2His2-type zinc fingers which, upon zinc metalation, recognize specific MRE sequences in the promoter region of MT genes triggering their expression. Cisplatin can efficiently react with protein metal binding sites featuring nucleophilic cysteine and/or histidine residues, including MTs and zinc fingers proteins, but the preferential reactivity towards specific targets with competing binding sites cannot be easily predicted. In this work, by in vitro competition reactions, we investigated the thermodynamic and kinetic preferential reactivity of cisplatin towards human Zn7MT-2, each of the six MTF-1 zinc fingers, and the entire human MTF-1 zinc finger domain. By spectroscopic, spectrometric, and electrophoretic mobility shift assays (EMSA), we demonstrated that cisplatin preferentially reacts with Zn7MT-2 to form Cys4-Pt(II) complexes, resulting in zinc release from MT-2. Zinc transfer from MT-2 to the MTF-1 triggers MTF-1 metalation, activation, and binding to target MRE sequences, as demonstrated by EMSA with DNA oligonucleotides. The cisplatin-dependent MT-mediated MTF-1 activation leading to apo-MT overexpression potentially establishes one of the molecular mechanisms underlying the development and potentiation of MT-mediated pre-target resistance.

Structural basis of ion uptake in copper-transporting P1B-type ATPases.

Copper is essential for living cells, yet toxic at elevated concentrations. Class 1B P-type (P1B-) ATPases are present in all kingdoms of life, facilitating cellular export of transition metals including copper. P-type ATPases follow an alternating access mechanism, with inward-facing E1 and outward-facing E2 conformations. Nevertheless, no structural information on E1 states is available for P1B-ATPases, hampering mechanistic understanding. Here, we present structures that reach 2.7 Å resolution of a copper-specific P1B-ATPase in an E1 conformation, with complementing data and analyses. Our efforts reveal a domain arrangement that generates space for interaction with ion donating chaperones, and suggest a direct Cu+ transfer to the transmembrane core. A methionine serves a key role by assisting the release of the chaperone-bound ion and forming a cargo entry site together with the cysteines of the CPC signature motif. Collectively, the findings provide insights into P1B-mediated transport, likely applicable also to human P1B-members.

Structures of Atm1 provide insight into [2Fe-2S] cluster export from mitochondria.

In eukaryotes, iron-sulfur clusters are essential cofactors for numerous physiological processes, but these clusters are primarily biosynthesized in mitochondria. Previous studies suggest mitochondrial ABCB7-type exporters are involved in maturation of cytosolic iron-sulfur proteins. However, the molecular mechanism for how the ABCB7-type exporters participate in this process remains elusive. Here, we report a series of cryo-electron microscopy structures of a eukaryotic homolog of human ABCB7, CtAtm1, determined at average resolutions ranging from 2.8 to 3.2 Å, complemented by functional characterization and molecular docking in silico. We propose that CtAtm1 accepts delivery from glutathione-complexed iron-sulfur clusters. A partially occluded state links cargo-binding to residues at the mitochondrial matrix interface that line a positively charged cavity, while the binding region becomes internalized and is partially divided in an early occluded state. Collectively, our findings substantially increase the understanding of the transport mechanism of eukaryotic ABCB7-type proteins.

Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu+ supplants entropically favoured Zn2+ to form Cu4 + clusters under physiological conditions.

Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d10 metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn2+ and Cu+ binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn2+ binding was measured by chelation titrations of Zn7MT-3, while Cu+ binding was measured by Zn2+ displacement from Zn7MT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant (K) and the change in enthalpy (ΔH) and entropy (ΔS) for these metal ions binding to MT-3. Zn2+ was also chelated from the individual α and ß domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn7MT-2 with Cu+ revealed that both MT isoforms have similar Cu+ affinities and binding thermodynamics, indicating that ΔH and ΔS are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu4 +-thiolate clusters when Cu+ displaces Zn2+ under physiological conditions. Comparison of the Zn2+ and Cu+ binding thermodynamics reveal that enthalpically-favoured Cu+, which forms Cu4 +-thiolate clusters, displaces the entropically-favoured Zn2+. These results provide a detailed thermodynamic analysis of d10 metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper.

PcoB is a defense outer membrane protein that facilitates cellular uptake of copper.

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical ß-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion-conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.

Evidence for a Long-Lived, Cu-Coupled and Oxygen-Inert Disulfide Radical Anion in the Assembly of Metallothionein-3 Cu(I)4-Thiolate Cluster.

The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I)4-Cys5-6 cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O2 and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn7MT-3 and Cu(II) to form Cu(I)4Zn(II)4MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430-450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu(II) leads to the formation of a redox-inert Cu(I)4-Cys5 cluster with short Cu-Cu distances (<2.8 Å), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu(II) reduction, generating the final Cu(I)4-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu(I), potentially explaining the observed DRA stability in the presence of O2. We thus provide evidence that the MT-3 Cu(I)4-Cys5 cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.

Structure and ion-release mechanism of PIB-4-type ATPases.

Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here, we present structures and complementary functional analyses of an archetypal PIB-4-ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy-metal-binding domains (HMBDs), and provide fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We also establish that the turnover of PIB-ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in for example drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.

Heavy metals such as zinc and cobalt are toxic at high levels, yet most organisms need tiny amounts for their cells to work properly. As a result, proteins studded through the cell membrane act as gatekeepers to finetune import and export. These proteins are central to health and disease; their defect can lead to fatal illnesses in humans, and they also help bacteria infect other organisms. Despite their importance, little is known about some of these metal-export proteins. This is particularly the case for P_IB-4-ATPases, a subclass found in plants and bacteria and which includes, for example, a metal transporter required for bacteria to cause tuberculosis. Intricate knowledge of the three-dimensional structure of these proteins would help to understand how they select metals, shuttle the compounds in and out of cells, and are controlled by other cellular processes. To reveal this three-dimensional organisation, Grønberg et al. used X-ray diffraction, where high-energy radiation is passed through crystals of protein to reveal the positions of atoms. They focused on a type of PIB-4-ATPases found in bacteria as an example. The work showed that the protein does not contain the metal-binding regions seen in other classes of metal exporters; however, it sports unique features that are crucial for metal transport such as an adapted pathway for the transport of zinc and cobalt across the membrane. In addition, Grønberg et al. tested thousands of compounds to see if they could block the activity of the protein, identifying two that could kill bacteria. This better understanding of how PIB-4-ATPases work could help to engineer plants capable of removing heavy metals from contaminated soils, as well as uncover new compounds to be used as antibiotics.

A single point mutation converts a proton-pumping rhodopsin into a red-shifted, turn-on fluorescent sensor for chloride.

The visualization of chloride in living cells with fluorescent sensors is linked to our ability to design hosts that can overcome the energetic penalty of desolvation to bind chloride in water. Fluorescent proteins can be used as biological supramolecular hosts to address this fundamental challenge. Here, we showcase the power of protein engineering to convert the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride in detergent micelles and in live Escherichia coli. This non-natural function was unlocked by mutating D121, which serves as the counterion to the protonated retinylidene Schiff base chromophore. Substitution from aspartate to valine at this position (D121V) creates a binding site for chloride. The binding of chloride tunes the pK a of the chromophore towards the protonated, fluorescent state to generate a pH-dependent response. Moreover, ion pumping assays combined with bulk fluorescence and single-cell fluorescence microscopy experiments with E. coli, expressing a GR1 fusion with a cyan fluorescent protein, show that GR1 does not pump ions nor sense membrane potential but instead provides a reversible, ratiometric readout of changes in extracellular chloride at the membrane. This discovery sets the stage to use natural and laboratory-guided evolution to build a family of rhodopsin-based fluorescent chloride sensors with improved properties for cellular applications and learn how proteins can evolve and adapt to bind anions in water.

Stabilization of supramolecular membrane protein-lipid bilayer assemblies through immobilization in a crystalline exoskeleton.

Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these complexes remains challenging because of their chemical composition, the hydrophobicity and structural instability of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid supramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework (ZIF) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF bio-composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embedded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional characterization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization.

A recombinant platform to characterize the role of transmembrane protein hTMEM205 in Pt(II)-drug resistance and extrusion.

Platinum-coordination complexes are among the most effective chemotherapeutic drugs used in clinics for the treatment of cancer. Despite their efficacy, cancer cells can develop drug resistance leading to treatment failure and relapse. Cellular uptake and extrusion of Pt(ii)-complexes mediated by transmembrane proteins are critical in controlling the intracellular concentration of Pt(ii)-drugs and in developing pre-target resistance. TMEM205 is a human transmembrane protein (hTMEM205) overexpressed in cancer cells that are resistant to cisplatin, but its molecular function underlying - resistance remains elusive. We developed a low-cost and high-throughput recombinant expression platform coupled to in vivo functional resistance assays to study the molecular mechanism by which the orphan hTMEM205 protects against Pt(ii)-complex toxicity. Based on the original observation by the Rosenberg group, which led to the discovery of cisplatin, we performed quantitative analysis of the effects of Pt(ii)-coordination complexes on cellular growth and filamentation in E. coli cells expressing hTMEM205. By coupling our methods with Pt quantification and cellular profiling in control and hTMEM205-expressing cells, we demonstrate that hTMEM205 mediates Pt(ii)-drug export selectively towards cisplatin and oxaliplatin but not carboplatin. By mutation analysis, we reveal that hTMEM205 recognizes and allows Pt(ii)-extrusion by a putative sulfur-based translocation mechanism, thereby resulting in pre-target resistance. Thus, hTMEM205 represents a new potential target that can be exploited to reduce cellular resistance towards Pt(ii)-drugs.

Membrane insertion exacerbates the α-Synuclein-Cu(II) dopamine oxidase activity: Metallothionein-3 targets and silences all α-synuclein-Cu(II) complexes.

Copper binding to α-synuclein (α-Syn), the major component of intracellular Lewy body inclusions in substantia nigra dopaminergic neurons, potentiate its toxic redox-reactivity and plays a detrimental role in the etiology of Parkinson disease (PD). Soluble α-synuclein-Cu(II) complexes possess dopamine oxidase activity and catalyze ROS production in the presence of biological reducing agents via Cu(II)/Cu(I) redox cycling. These metal-centered redox reactivities harmfully promote the oxidation and oligomerization of α-Syn. While this chemistry has been investigated on recombinantly expressed soluble α-Syn, in vivo, α-Syn is acetylated at its N-terminus and is present in equilibrium between soluble and membrane-bound forms. This post-translational modification and membrane-binding alter the Cu(II) coordination environment and binding modes and are expected to affect the α-Syn-Cu(II) reactivity. In this work, we first investigated the reactivity of acetylated and membrane-bound complexes, and subsequently addressed whether the brain metalloprotein Zn7-metallothionein-3 (Zn7MT-3) possesses a multifaceted-role in targeting these aberrant copper interactions and consequent reactivity. Through biochemical characterization of the reactivity of the non-acetylated/N-terminally acetylated soluble or membrane-bound α-Syn-Cu(II) complexes towards dopamine, oxygen, and ascorbate, we reveal that membrane insertion dramatically exacerbates the catechol oxidase-like reactivity of α-Syn-Cu(II) as a result of a change in the Cu(II) coordination environment, thereby potentiating its toxicity. Moreover, we show that Zn7MT-3 can efficiently target all α-Syn-Cu(II) complexes through Cu(II) removal, preventing their deleterious redox activities. We demonstrate that the Cu(II) reduction by the thiolate ligands of Zn7MT-3 and the formation of Cu(I)4Zn4MT-3 featuring an unusual redox-inert Cu(I)4-thiolate cluster is the molecular mechanism responsible for the protective effect exerted by MT-3 towards α-Syn-Cu(II). This work provides the molecular basis for new therapeutic interventions to control the deleterious bioinorganic chemistry of α-Syn-Cu(II).

Transmembrane Cu(I) P-type ATPase pumps are electrogenic uniporters.

Cu(i) P-type ATPases are transmembrane primary active ion pumps that catalyze the extrusion of copper ions across cellular membranes. Their activity is critical in controlling copper levels in all kingdoms of life. Biochemical and structural characterization established the structural framework by which Cu-pumps perform their function. However, the details of the overall mechanism of transport (uniporter vs. cotransporter) and electrogenicity still remain elusive. In this work, we developed a platform to reconstitute the model Cu(i)-pump from E. coli (EcCopA) in artificial lipid bilayer small unilamellar vesicles (SUVs) to quantitatively characterize the metal substrate, putative counter-ions and charge translocation. By encapsulating in the liposome lumen fluorescence detector probes (CTAP-3, pyranine and oxonol VI) responsive to diverse stimuli (Cu(i), pH and membrane potential), we correlated substrate, secondary-ion translocation and charge movement events in EcCopA proteoliposomes. This platform centered on multiple fluorescence reporters allowed study of the mechanism and translocation kinetic parameters in real-time for wild-type EcCopA and inactive mutants. The maximal initial Cu(i) transport rate of 165 nmol Cu(i) mg-1 min-1 and KM, Cu(I) = 0.15 ± 0.07 µM was determined with this analysis. We reveal that Cu(i) pumps are primary-active uniporters and electrogenic. The Cu(i) translocation cycle does not require proton counter-transport resulting in electrogenic generation of transmembrane potential upon translocation of one Cu(i) per ATP hydrolysis cycle. Thus, mechanistic differences between Cu(i) pumps and other better characterized P-type ATPases are discussed. The platform opens the venue to study translocation events and mechanisms of transport in other transition metal P-type ATPase pumps.

The ABC exporter IrtAB imports and reduces mycobacterial siderophores.

Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores that scavenge iron from host proteins1. M. tuberculosis produces two classes of siderophore, lipid-bound mycobactin and water-soluble carboxymycobactin2,3. Functional studies have revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ATP binding cassette (ABC) transporter IrtAB4, which features an additional cytoplasmic siderophore interaction domain5. However, the predicted ABC exporter fold of IrtAB is seemingly contradictory to its import function. Here we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the siderophore interaction domain to facilitate iron release. Structure determination by X-ray crystallography and cryo-electron microscopy not only confirms that IrtAB has an ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB that result in a partially collapsed inward-facing substrate-binding cavity. The siderophore interaction domain is positioned in close proximity to the inner membrane leaflet, enabling the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB has a preference for mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore-import machinery in mycobacteria.

Mutations in Superoxide Dismutase 1 (Sod1) Linked to Familial Amyotrophic Lateral Sclerosis Can Disrupt High-Affinity Zinc-Binding Promoted by the Copper Chaperone for Sod1 (Ccs).

Zinc (II) ions (hereafter simplified as zinc) are important for the structural and functional activity of many proteins. For Cu, Zn superoxide dismutase (Sod1), zinc stabilizes the native structure of each Sod1 monomer, promotes homo-dimerization and plays an important role in activity by "softening" the active site so that copper cycling between Cu(I) and Cu(II) can rapidly occur. Previously, we have reported that binding of Sod1 by its copper chaperone (Ccs) stabilizes a conformation of Sod1 that promotes site-specific high-affinity zinc binding. While there are a multitude of Sod1 mutations linked to the familial form of amyotrophic lateral sclerosis (fALS), characterizations by multiple research groups have been unable to realize strong commonalities among mutants. Here, we examine a set of fALS-linked Sod1 mutations that have been well-characterized and are known to possess variation in their biophysical characteristics. The zinc affinities of these mutants are evaluated here for the first time and then compared with the previously established value for wild-type Sod1 zinc affinity. Ccs does not have the same ability to promote zinc binding to these mutants as it does for the wild-type version of Sod1. Our data provides a deeper look into how (non)productive Sod1 maturation by Ccs may link a diverse set of fALS-Sod1 mutations.

The Glutathione/Metallothionein System Challenges the Design of Efficient O2 -Activating Copper Complexes.

Copper complexes are of medicinal and biological interest, including as anticancer drugs designed to cleave intracellular biomolecules by O2 activation. To exhibit such activity, the copper complex must be redox active and resistant to dissociation. Metallothioneins (MTs) and glutathione (GSH) are abundant in the cytosol and nucleus. Because they are thiol-rich reducing molecules with high CuI affinity, they are potential competitors for a copper ion bound in a copper drug. Herein, we report the investigation of a panel of CuI /CuII complexes often used as drugs, with diverse coordination chemistries and redox potentials. We evaluated their catalytic activity in ascorbate oxidation based on redox cycling between CuI and CuII , as well as their resistance to dissociation or inactivation under cytosolically relevant concentrations of GSH and MT. O2 -activating CuI /CuII complexes for cytosolic/nuclear targets are generally not stable against the GSH/MT system, which creates a challenge for their future design.

IroT/MavN Is a Legionella Transmembrane Fe(II) Transporter: Metal Selectivity and Translocation Kinetics Revealed by in Vitro Real-Time Transport.

In intravacuolar pathogens, iron is essential for growth and virulence. In Legionella pneumophila, a putative transmembrane protein inserted on the surface of the host pathogen-containing vacuole, IroT/MavN, facilitates intravacuolar iron acquisition from the host by an unknown mechanism, bypassing the problem of Fe(III) insolubility and mobilization. We developed a platform for purification and reconstitution of IroT in artificial lipid bilayer vesicles (proteoliposomes). By encapsulating the fluorescent reporter probe Fluozin-3, we reveal, by real-time metal transport assays, that IroT is a high-affinity iron transporter selective for Fe(II) over other essential transition metals. Mutational analysis reveals important residues in the transmembrane helices, soluble domains, and loops important for substrate recognition and translocation. The work establishes the substrate transport properties in a novel transporter family important for iron acquisition at the host-pathogen intravacuolar interface and provides chemical tools for a comparative investigation of the translocation properties in other iron transporter families.