Unraveling Copper Exchange in the Atox1-Cu(I)-Mnk1 Heterodimer: A Simulation Approach.

Copper, an essential metal for various cellular processes, requires tight regulation to prevent cytotoxicity. Intracellular pathways crucial for maintaining optimal copper levels involve soluble and membrane transporters, namely, metallochaperones and P-type ATPases, respectively. In this study, we used a simulation workflow based on free-energy perturbation (FEP) theory and parallel bias metadynamics (PBMetaD) to predict the Cu(I) exchange mechanism between the human Cu(I) chaperone, Atox1, and one of its two physiological partners, ATP7A. ATP7A, also known as the Menkes disease protein, is a transmembrane protein and one of the main copper-transporting ATPases. It pumps copper into the trans-Golgi network for the maturation of cuproenzymes and is also essential for the efflux of excess copper across the plasma membrane. In this analysis, we utilized the nuclear magnetic resonance (NMR) structure of the Cu(I)-mediated complex between Atox1 and the first soluble domain of the Menkes protein (Mnk1) as a starting point. Independent free-energy simulations were conducted to investigate the dissociation of both Atox1 and Mnk1. The calculations revealed that the two dissociations require free energy values of 6.3 and 6.2 kcal/mol, respectively, following a stepwise dissociation mechanism.

Dynamical interplay between the human high-affinity copper transporter hCtr1 and its cognate metal ion.

Abnormal cellular copper levels have been clearly implicated in genetic diseases, cancer, and neurodegeneration. Ctr1, a high-affinity copper transporter, is a homotrimeric integral membrane protein that provides the main route for cellular copper uptake. Together with a sophisticated copper transport system, Ctr1 regulates Cu(I) metabolism in eukaryotes. Despite its pivotal role in normal cell function, the molecular mechanism of copper uptake and transport via Ctr1 remains elusive. In this study, electron paramagnetic resonance (EPR), UV-visible spectroscopy, and all-atom simulations were employed to explore Cu(I) binding to full-length human Ctr1 (hCtr1), thereby elucidating how metal binding at multiple distinct sites affects the hCtr1 conformational dynamics. We demonstrate that each hCtr1 monomer binds up to five Cu(I) ions and that progressive Cu(I) binding triggers a marked structural rearrangement in the hCtr1 C-terminal region. The observed Cu(I)-induced conformational remodeling suggests that the C-terminal region may play a dual role, serving both as a channel gate and as a shuttle mediating the delivery of copper ions from the extracellular hCtr1 selectivity filter to intracellular metallochaperones. Our findings thus contribute to a more complete understanding of the mechanism of hCtr1-mediated Cu(I) uptake and provide a conceptual basis for developing mechanism-based therapeutics for treating pathological conditions linked to de-regulated copper metabolism.

Identification and molecular characterization of the high-affinity copper transporters family in Solanum lycopersicum.

Copper (Cu) plays a key role as cofactor in the plant proteins participating in essential cellular processes, such as electron transport and free radical scavenging. Despite high-affinity Cu transporters (COPTs) being key participants in Cu homeostasis maintenance, very little is known about COPTs in tomato (Solanum lycopersicum) even though it is the most consumed fruit worldwide and this crop is susceptible to suboptimal Cu conditions. In this study, a six-member family of COPT (SlCOPT1-6) was identified and characterized. SlCOPTs have a conserved architecture consisting of three transmembrane domains and ß-strains. However, the presence of essential methionine residues, a methionine-enriched amino-terminal region, an Mx3Mx12Gx3G Cu-binding motif and a cysteine rich carboxy-terminal region, all required for their functionality, is more variable among members. Accordingly, functional complementation assays in yeast indicate that SlCOPT1 and SlCOPT2 are able to transport Cu inside the cell, while SlCOPT3 and SlCOPT5 are only partially functional. In addition, protein interaction network analyses reveal the connection between SlCOPTs and Cu PIB-type ATPases, other metal transporters, and proteins related to the peroxisome. Gene expression analyses uncover organ-dependency, fruit vasculature tissue specialization and ripening-dependent gene expression profiles, as well as different response to Cu deficiency or toxicity in an organ-dependent manner.

Molecular characterization of the COPT/Ctr-type copper transporter family under heavy metal stress in alfalfa.

In nature, heavy metals significantly affect crop growth and quality. Among various heavy metals, copper (Cu) is both essential and toxic to plants depending on the concentration and complex homeostatic networks. The Cu transporter family (COPT) plays important roles in Cu homeostasis, including absorption, transportation, and growth in plants; however, this gene family is still poorly understood in alfalfa (Medicago sativa L.). In this study, a total of 12 MsCOPTs were identified and characterized. Based on the conserved motif and phylogenetic analysis, MsCOPTs could be divided into four subgroups (A1, A2, A3, and B). Gene structure, chromosomal location, and synteny analyses of MsCOPTs showed that segmental and tandem duplications likely contributed to their evolution. Tissue-specific expression analysis of MsCOPT genes indicated diverse spatiotemporal expression patterns. Most MsCOPT genes had high transcription levels in roots and nodules, indicating that these genes may play vital roles in the absorption and transport of Cu through root. The complementary heterologous expression function of yeast once again indicates that root-specific COPT can supplement the growth of defective yeast strains on YPEG medium, suggesting that these genes are Cu transporters. In summary, for the first time, our research identified COPT family genes at the whole-genome level to provide guidance for effectively improving the problem of Cu deficiency in the grass-livestock chain and provide theoretical support for the subsequent development of grass and animal husbandry.

Expression and Purification of the VpDef Defensin in Escherichia coli using the Small Metal-Binding Proteins CusF3H+ and SmbP.

BACKGROUND: The heterologous production of antimicrobial peptides in bacterial models can produce insoluble proteins due to the lack of proper folding. Fusion proteins have been used to increase the expression and solubility of these types of proteins with varying degrees of success. OBJECTIVES: Here, we demonstrate the use of the small metal-binding proteins CusF3H+ (9.9kDa) and SmbP (9.9kDa) as fusion partners for the soluble expression of the bioactive antimicrobial peptide VpDef(6.9 kDa) in Escherichia coli. METHODS: The recombinant VpDef (rVpDef) peptide was expressed as a translational fusion with CusF3H+ and SmbP in Escherichia coli SHuffle under different small-scale culture conditions. The best conditions were applied to 1-liter cultures, with subsequent purification of the recombinant protein through IMAC chromatography. The recombinant protein was digested using enterokinase to liberate the peptide from the fusion protein, and a second IMAC chromatography step removed the fusion protein. The purified peptide was tested against two Gram-positive and two Gram-negative bacteria. RESULTS: The use either of CusF3H+ or of SmbP results in recombinant proteins that are found in the soluble fraction of the bacterial lysate; these recombinant proteins are easily purified through IMAC chromatography, and rVpDef is readily separated following enterokinase treatment. The purified rVpDef peptide exhibits antimicrobial properties against both Gram-positive and Gram-negative. CONCLUSION: Use of the fusion proteins CusF3H+ and SmbP results in production of a soluble recombinant protein containing the antimicrobial peptide rVpDef that is correctly folded and that retains its antimicrobial properties once purified.

Expression and Purification of Recombinant Proteins in Escherichia coli Tagged with the Metal-Binding Proteins SmbP and CusF3H.

The bacterium Escherichia coli is still considered the first option as a microbial cell factory for recombinant protein production, and affinity chromatography is by far the preferred technique for initial purification after protein expression and cell lysis. In this chapter, we describe the methodology to express and purify recombinant proteins in E. coli tagged with the first two metal-binding proteins proposed as fusion partners. They are the small metal-binding protein SmbP and a mutant of the copper resistance protein CusF3H+. There are several advantages of using them as protein tags: they prevent the formation of inclusion bodies by increasing solubility of the target proteins, they enable purification by immobilized metal-affinity chromatography using Ni(II) ions with high purity, and because of their low molecular weights, excellent final yields are obtained for the target proteins after cleavage and removal of the protein tag. Here we also describe the protocol for the production of proteins in the periplasm of E. coli tagged with two SmbP variants that include the PelB or the TorA signal sequences for transport via the Sec or the Tat pathway, respectively. Based on these methods, we consider CusF3H+ and SmbP excellent alternatives as fusion proteins for the production of recombinant proteins in E. coli.

Structural and Functional Diversity Among the Members of CTR, the Membrane Copper Transporter Family.

Copper is crucial for carrying out normal physiological functions in all higher life forms. Copper Transporter 1 (CTR1) is the high-affinity copper importer found in all eukaryotic organisms. The copper transporter family primarily comprises ~ six members (CTR1-6) and the related members share high sequence homology with CTR. However, with the exception of CTR1, not all six CTRs are present in every organism. Despite having a simple trimeric channel structure, CTR1 and other members exhibit some unique regulatory properties. In the present review, we attempt to understand the diversity and similarity of regulation and functioning of the members of this copper transporter family.

An EPR Study on the Interaction between the Cu(I) Metal Binding Domains of ATP7B and the Atox1 Metallochaperone.

Copper's essentiality and toxicity mean it requires a sophisticated regulation system for its acquisition, cellular distribution and excretion, which until now has remained elusive. Herein, we applied continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy in solution to resolve the copper trafficking mechanism in humans, by considering the route travelled by Cu(I) from the metallochaperone Atox1 to the metal binding domains of ATP7B. Our study revealed that Cu(I) is most likely mediated by the binding of the Atox1 monomer to metal binding domain 1 (MBD1) and MBD4 of ATP7B in the final part of its extraction pathway, while the other MBDs mediate this interaction and participate in copper transfer between the various MBDs to the ATP7B membrane domain. This research also proposes that MBD1-3 and MBD4-6 act as two independent units.

Arabidopsis thaliana Hcc1 is a Sco-like metallochaperone for CuA assembly in Cytochrome c Oxidase.

The assembly of the CuA site in Cytochrome c Oxidase (COX) is a critical step for aerobic respiration in COX-dependent organisms. Several gene products have been associated with the assembly of this copper site, the most conserved of them belonging to the Sco family of proteins, which have been shown to perform different roles in different organisms. Plants express two orthologs of Sco proteins: Hcc1 and Hcc2. Hcc1 is known to be essential for plant development and for COX maturation, but its precise function has not been addressed until now. Here, we report the biochemical, structural and functional characterization of Arabidopsis thaliana Hcc1 protein (here renamed Sco1). We solved the crystal structure of the Cu+1 -bound soluble domain of this protein, revealing a tri coordinated environment involving a CxxxCxn H motif. We show that AtSco1 is able to work as a copper metallochaperone, inserting two Cu+1 ions into the CuA site in a model of CoxII. We also show that AtSco1 does not act as a thiol-disulfide oxido-reductase. Overall, this information sheds new light on the biochemistry of Sco proteins, highlighting the diversity of functions among them despite their high structural similarities. DATABASE: PDB entry 6N5U (Crystal structure of Arabidopsis thaliana ScoI with copper bound).

Inhibiting the copper efflux system in microbes as a novel approach for developing antibiotics.

Five out of six people receive at least one antibiotic prescription per year. However, the ever-expanding use of antibiotics in medicine, agriculture, and food production has accelerated the evolution of antibiotic-resistant bacteria, which, in turn, made the development of novel antibiotics based on new molecular targets a priority in medicinal chemistry. One way of possibly combatting resistant bacterial infections is by inhibiting the copper transporters in prokaryotic cells. Copper is a key element within all living cells, but it can be toxic in excess. Both eukaryotic and prokaryotic cells have developed distinct copper regulation systems to prevent its toxicity. Therefore, selectively targeting the prokaryotic copper regulation system might be an initial step in developing next-generation antibiotics. One such system is the Gram-negative bacterial CusCFBA efflux system. CusB is a key protein in this system and was previously reported to play an important role in opening the channel for efflux via significant structural changes upon copper binding while also controlling the assembly and disassembly process of the entire channel. In this study, we aimed to develop novel peptide copper channel blockers, designed by in silico calculations based on the structure of CusB. Using a combination of magnetic resonance spectroscopy and various biochemical methods, we found a lead peptide that promotes copper-induced cell toxicity. Targeting copper transport in bacteria has not yet been pursued as an antibiotic mechanism of action. Thus, our study lays the foundation for discovering novel antibiotics.

Catalytic M Center of Copper Monooxygenases Probed by Rational Design. Effects of Selenomethionine and Histidine Substitution on Structure and Reactivity.

The M centers of the mononuclear monooxygenases peptidylglycine monooxygenase (PHM) and dopamine ß-monooxygenase bind and activate dioxygen en route to substrate hydroxylation. Recently, we reported the rational design of a protein-based model in which the CusF metallochaperone was repurposed via a His to Met mutation to act as a structural and spectroscopic biomimic. The PHM M site exhibits a number of unusual attributes, including a His2Met ligand set, a fluxional Cu(I)-S(Met) bond, tight binding of exogenous ligands CO and N3-, and complete coupling of oxygen reduction to substrate hydroxylation even at extremely low turnover rates. In particular, mutation of the Met ligand to His completely eliminates the catalytic activity despite the propensity of CuI-His3 centers to bind and activate dioxygen in other metalloenzyme systems. Here, we further develop the CusF-based model to explore methionine variants in which Met is replaced by selenomethionine (SeM) and histidine. We examine the effects on coordinate structure and exogenous ligand binding via X-ray absorption spectroscopy and electron paramagnetic resonance and probe the consequences of mutations on redox chemistry via studies of the reduction by ascorbate and oxidation via molecular oxygen. The M-site model is three-coordinate in the Cu(I) state and binds CO to form a four-coordinate carbonyl. In the oxidized forms, the coordination changes to tetragonal five-coordinate with a long axial Met ligand that like the enzymes is undetectable at either the Cu or Se K edges. The EXAFS data at the Se K edge of the SeM variant provide unique information about the nature of the Cu-methionine bond that is likewise weak and fluxional. Kinetic studies document the sluggish reactivity of the Cu(I) complexes with molecular oxygen and rapid rates of reduction of the Cu(II) complexes by ascorbate, indicating a remarkable stability of the Cu(I) state in all three derivatives. The results show little difference between the Met ligand and its SeM and His congeners and suggest that the Met contributes to catalysis in ways that are more complex than simple perturbation of the redox chemistry. Overall, the results stimulate a critical re-examination of the canonical reaction mechanisms of the mononuclear copper monooxygenases.

Interaction between Copper Chaperone Atox1 and Parkinson's Disease Protein α-Synuclein Includes Metal-Binding Sites and Occurs in Living Cells.

Alterations in copper ion homeostasis appear coupled to neurodegenerative disorders, but mechanisms are unknown. The cytoplasmic copper chaperone Atox1 was recently found to inhibit amyloid formation in vitro of α-synuclein, the amyloidogenic protein in Parkinson's disease. As α-synuclein may have copper-dependent functions, and free copper ions promote α-synuclein amyloid formation, it is important to characterize the Atox1 interaction with α-synuclein on a molecular level. Here we applied solution-state nuclear magnetic resonance spectroscopy, with isotopically labeled α-synuclein and Atox1, to define interaction regions in both proteins. The α-synuclein interaction interface includes the whole N-terminal part up to Gln24; in Atox1, residues around the copper-binding cysteines (positions 11-16) are mostly perturbed, but additional effects are also found for residues elsewhere in both proteins. Because α-synuclein is N-terminally acetylated in vivo, we established that Atox1 also inhibits amyloid formation of this variant in vitro, and proximity ligation in human cell lines demonstrated α-synuclein-Atox1 interactions in situ. Thus, this interaction may provide the direct link between copper homeostasis and amyloid formation in vivo.

Oxidation of Human Copper Chaperone Atox1 and Disulfide Bond Cleavage by Cisplatin and Glutathione.

Cancer cells cope with high oxidative stress levels, characterized by a shift toward the oxidized form (GSSG) of glutathione (GSH) in the redox couple GSSG/2GSH. Under these conditions, the cytosolic copper chaperone Atox1, which delivers Cu(I) to the secretory pathway, gets oxidized, i.e., a disulfide bond is formed between the cysteine residues of the Cu(I)-binding CxxC motif. Switching to the covalently-linked form, sulfur atoms are not able to bind the Cu(I) ion and Atox1 cannot play an antioxidant role. Atox1 has also been implicated in the resistance to platinum chemotherapy. In the presence of excess GSH, the anticancer drug cisplatin binds to Cu(I)-Atox1 but not to the reduced apoprotein. With the aim to investigate the interaction of cisplatin with the disulfide form of the protein, we performed a structural characterization in solution and in the solid state of oxidized human Atox1 and explored its ability to bind cisplatin under conditions mimicking an oxidizing environment. Cisplatin targets a methionine residue of oxidized Atox1; however, in the presence of GSH as reducing agent, the drug binds irreversibly to the protein with ammine ligands trans to Cys12 and Cys15. The results are discussed with reference to the available literature data and a mechanism is proposed connecting platinum drug processing to redox and copper homeostasis.

Potential role of serotonin as a biological reductant associated with copper transportation.

Serotonin (5-HT) is a neurotransmitter that is derived from tryptophan. Owing to a hydroxyl group attached to the indole nucleus, 5-HT exhibits a considerably higher redox activity than tryptophan. To gain insight into the biological relevance of the redox activity of 5-HT, the effect of Cu(I)-binding ligands on the 5-HT-mediated copper reduction was investigated. The d-d transition band of Cu(II) complexed with glycine [Cu(II)-Gly2] was not affected by addition of 5-HT alone but was diminished when a thioether-containing compound coexists with 5-HT. Concomitant with disappearance of the d-d transition band of Cu(II)-Gly2, the π-π* transition band of 5-hydroxyindole of 5-HT exhibits a red-shift which is consistently explained by oxidation of 5-HT and subsequent formation of a dimeric species. The redox reactions between 5-HT and copper are also accelerated by a peptide composed of a methionine (Met)-rich region in the extracellular domain of an integral membrane protein, copper transporter 1 (Ctr1). Since Ctr1 transports copper across the plasma membrane with specificity for Cu(I), reduction of extracellular Cu(II) to Cu(I) is required for copper uptake by Ctr1. Metalloreductases that can donate Cu(I) for Ctr1 have been identified in yeast but not yet been found in mammals. The results of this study indicate that the Met-rich region in the N-terminal extracellular domain of Ctr1 promotes the 5-HT-mediated Cu(II) reduction in order to acquire Cu(I) via a non-enzymatic process.

Copper trafficking in eukaryotic systems: current knowledge from experimental and computational efforts.

Copper plays a vital role in fundamental cellular functions, and its concentration in the cell must be tightly regulated, as dysfunction of copper homeostasis is linked to severe neurological diseases and cancer. This review provides a compendium of current knowledge regarding the mechanism of copper transfer from the blood system to the Golgi apparatus; this mechanism involves the copper transporter hCtr1, the metallochaperone Atox1, and the ATPases ATP7A/B. We discuss key insights regarding the structural and functional properties of the hCtr1-Atox1-ATP7B cycle, obtained from diverse studies relying on distinct yet complementary biophysical, biochemical, and computational methods. We further address the mechanistic aspects of the cycle that continue to remain elusive. These knowledge gaps must be filled in order to be able to harness our understanding of copper transfer to develop therapeutic approaches with the capacity to modulate copper metabolism.