Human Copper Transporter Research Articles

Predicting Conformational Ensembles of Intrinsically Disordered Proteins: From Molecular Dynamics to Machine Learning.

Intrinsically disordered proteins and regions (IDP/IDRs) are ubiquitous across all domains of life. Characterized by a lack of a stable tertiary structure, IDP/IDRs populate a diverse set of transiently formed structural states that can promiscuously adapt upon binding with specific interaction partners and/or certain alterations in environmental conditions. This malleability is foundational for their role as tunable interaction hubs in core cellular processes such as signaling, transcription, and translation. Tracing the conformational ensemble of an IDP/IDR and its perturbation in response to regulatory cues is thus paramount for illuminating its function. However, the conformational heterogeneity of IDP/IDRs poses several challenges. Here, we review experimental and computational methods devised to disentangle the conformational landscape of IDP/IDRs, highlighting recent computational advances that permit proteome-wide scans of IDP/IDRs conformations. We briefly evaluate selected computational methods using the disordered N-terminal of the human copper transporter 1 as a test case and outline further challenges in IDP/IDRs ensemble prediction.

64Cu]Copper chloride PET-CT: a comparative evaluation of fasting and non-fasting states in patients of prostate carcinoma.

Altered copper metabolism in cancer has been linked to increased intracellular copper uptake mediated by human copper transporter 1, with [64Cu]Cu2+ as a potential biomarker for cancer theranostics. [64Cu]CuCl2 PET-CT though explored in various malignancies, a lack of standardized protocol exists, particularly regarding fasting status before imaging. This analysis aimed to evaluate the requirement of fasting for [64Cu]CuCl2 PET-CT along with temporal changes in physiological organ uptake in delayed scans. A total of 26 patients of prostate carcinoma who underwent [64Cu]CuCl2 PET-CT imaging were divided into two groups: (1) nonfasting (n = 12) and (2) fasting (n = 14). The nonfasting group received an average dose of 350 MBq, while the fasting group received 300 MBq of [64Cu]CuCl2, and PET-CT images acquired approximately 60-90 min (1 h image) and 3-3.5 h (delayed image) after intravenous injection of the tracer. An experienced nuclear medicine physician evaluated the images for qualitative assessment between the groups. Multiple spherical regions of interest were placed at sites of physiological organ uptake of the tracer and over the diseased lesions to measure the mean SUVmax. No significant difference was observed in the qualitative assessment of the images between the two groups (except for a slight predilection towards more hepatic tracer retention observed in the fasting group), including in the delayed images. The liver demonstrated the highest tracer uptake in all patients, with a mean SUVmax of 21.5 in the fasting group and 19.7 in the nonfasting group, showing no significant difference (P = 0.32). The kidneys, intestines, and salivary glands also showed similar trends of tracer uptake in both groups. The study illustrated that the fasting or nonfasting status did not affect image quality or semiquantitative measurements significantly in physiological organs and diseased lesions in patients with carcinoma prostate.

Exploration of the Potential Role of Serum Albumin in the Delivery of Cu(I) to Ctr1.

Human serum albumin (HSA) is the major copper (Cu) carrier in blood. The majority of previous studies that have investigated Cu interactions with HSA have focused primarily on the Cu(II) oxidation state. Yet, cellular Cu uptake by the human copper transport protein (Ctr1), a plasma membrane-embedded protein responsible for Cu uptake into cells, requires Cu(I). Recent in vitro work has determined that reducing agents, such as the ascorbate present in blood, are sufficient to reduce the Cu(II)HSA complex to form Cu(I)HSA and that Cu(I) is bound to HSA with pM affinity. The biological accessibility of Cu(I)HSA suggests that HSA-bound Cu(I) may be an unappreciated form of Cu cargo and a key player in extracellular Cu trafficking. To better understand Cu trafficking by HSA, we sought to investigate the exchange of Cu(I) from HSA to a model peptide of the Cu-binding ectodomain of Ctr1. In this study, we used X-ray absorption near-edge spectroscopy to show that Cu(I) becomes more highly coordinated as increasing amounts of the Ctr1-14 model peptide are added to a solution of Cu(I)HSA. Extended X-ray absorption fine structure (EXAFS) spectroscopy was used to further characterize the interaction of Cu(I)HSA with Ctr1-14 by determining the ligands coordinating Cu(I) and their bond lengths. The EXAFS data support that some Cu(I) likely undergoes complete transfer from HSA to Ctr1-14. This finding of HSA interacting with and releasing Cu(I) to an ectodomain model peptide of Ctr1 suggests a mechanism by which HSA delivers Cu(I) to cells under physiological conditions.

Properties of recombinant extracellular N-terminal domain of human high-affinity copper transporter 1 (hNdCTR1) and its interactions with Cu(II) and Ag(I) ions.

High-affinity copper transporter 1 (CTR1) is a key link in the transfer of copper (Cu) from the extracellular environment to the cell. Violation in the control system of its expression, or mutations in this gene, cause a global copper imbalance. However, the mechanism of copper transfer via CTR1 remains unclear. It has been shown that transformed bacteria synthesizing the fused GB1-NdCTR become resistant to toxic silver ions. According to UV-Vis spectrophotometry and isothermal titration calorimetry, electrophoretically pure GB1-NdCTR specifically and reversibly binds copper and silver ions, and binding is associated with aggregation. Purified NdCTR1 forms SDS-resistant oligomers. The link between nontrivial properties of NdCTR1 and copper import mechanism from extracellular space, as well as potential chelating properties of NdCTR1, are discussed.

Targeting Copper in Cancer Imaging and Therapy: A New Theragnostic Agent

Copper is required for cancer cell proliferation and tumor angiogenesis. Copper-64 radionuclide (64Cu), a form of copper chloride (64CuCl2), is rapidly emerging as a diagnostic PET/CT tracer in oncology. It may also represent an interesting alternative to gallium-68 (68Ga) as a radionuclide precursor for labelling radiopharmaceuticals used to investigate neuroendocrine tumors and prostate cancer. This emerging interest is also related to the nuclear properties of 64CuCl2 that make it an ideal theragnostic nuclide. Indeed, 64CuCl2 emits β+ and β- particles together with high-linear-energy-transfer Auger electrons, suggesting the therapeutic potential of 64CuCl2 for the radionuclide cancer therapy of copper-avid tumors. Recently, 64CuCl2 was successfully used to image prostate cancer, bladder cancer, glioblastoma multiforme (GBM), and non-small cell lung carcinoma in humans. Copper cancer uptake was related to the expression of human copper transport 1 (hCTR1) on the cancer cell surface. Biodistribution, toxicology and radiation safety studies showed its radiation and toxicology safety. Based on the findings from the preclinical research studies, 64CuCl2 PET/CT also holds potential for the diagnostic imaging of human hepatocellular carcinoma (HCC), malignant melanoma, and the detection of the intracranial metastasis of copper-avid tumors based on the low physiological background of radioactive copper uptake in the brain.

EPR Spectroscopy Provides New Insights into Complex Biological Reaction Mechanisms.

In the last 20 years, the use of electron paramagnetic resonance (EPR) has made a pronounced and lasting impact in the field of structural biology. The advantage of EPR spectroscopy over other structural techniques is its ability to target even minor conformational changes in any biomolecule or macromolecular complex, independent of its size or complexity, or whether it is in solution or in the cell during a biological or chemical reaction. Here, we focus on the use of EPR spectroscopy to study transmembrane transport and transcription mechanisms. We discuss experimental and analytical concerns when referring to studies of two biological reaction mechanisms, namely, transfer of copper ions by the human copper transporter hCtr1 and the mechanism of action of the Escherichia coli copper-dependent transcription factor CueR. Last, we elaborate on future avenues in the field of EPR structural biology.

Copper binding leads to increased dynamics in the regulatory N-terminal domain of full-length human copper transporter ATP7B.

ATP7B is a human copper-transporting P1B-type ATPase that is involved in copper homeostasis and resistance to platinum drugs in cancer cells. ATP7B consists of a copper-transporting core and a regulatory N-terminal tail that contains six metal-binding domains (MBD1-6) connected by linker regions. The MBDs can bind copper, which changes the dynamics of the regulatory domain and activates the protein, but the underlying mechanism remains unknown. To identify possible copper-specific structural dynamics involved in transport regulation, we constructed a model of ATP7B spanning the N-terminal tail and core catalytic domains and performed molecular dynamics (MD) simulations with (holo) and without (apo) copper ions bound to the MBDs. In the holo protein, MBD2, MBD3 and MBD5 showed enhanced mobilities, which resulted in a more extended N-terminal regulatory region. The observed separation of MBD2 and MBD3 from the core protein supports a mechanism where copper binding activates the ATP7B protein by reducing interactions among MBD1-3 and between MBD1-3 and the core protein. We also observed an increased interaction between MBD5 and the core protein that brought the copper-binding site of MBD5 closer to the high-affinity internal copper-binding site in the core protein. The simulation results assign specific, mechanistic roles to the metal-binding domains involved in ATP7B regulation that are testable in experimental settings.

Multinuclear Metal-Binding Ability of the N-Terminal Region of Human Copper Transporter Ctr1: Dependence Upon pH and Metal Oxidation State.

The 14mer peptide corresponding to the N-terminal region of human copper transporter Ctr1 was used to investigate the intricate mechanism of metal binding to this plasma membrane permease responsible for copper import in eukaryotic cells. The peptide contains a high-affinity ATCUN Cu(II)/Ni(II)-selective motif, a methionine-only MxMxxM Cu(I)/Ag(I)-selective motif and a double histidine HH(M) motif, which can bind both Cu(II) and Cu(I)/Ag(I) ions. Using a combination of NMR spectroscopy and electrospray mass spectrometry, clear evidence was gained that the Ctr1 peptide, at neutral pH, can bind one or two metal ions in the same or different oxidation states. Addition of ascorbate to a neutral solution containing Ctr11-14 and Cu(II) in 1:1 ratio does not cause an appreciable reduction of Cu(II) to Cu(I), which is indicative of a tight binding of Cu(II) to the ATCUN motif. However, by lowering the pH to 3.5, the Cu(II) ion detaches from the peptide and becomes susceptible to reduction to Cu(I) by ascorbate. It is noteworthy that at low pH, unlike Cu(II), Cu(I) stably binds to methionines of the peptide. This redox reaction could take place in the lumen of acidic organelles after Ctr1 internalization. Unlike Ctr11-14-Cu(II), bimetallic Ctr11-14-2Cu(II) is susceptible to partial reduction by ascorbate at neutral pH, which is indicative of a lower binding affinity of the second Cu(II) ion. The reduced copper remains bound to the peptide, most likely to the HH(M) motif. By lowering the pH to 3.5, Cu(I) shifts from HH(M) to methionine-only coordination, an indication that only the pH-insensitive methionine motif is competent for metal binding at low pH. The easy interconversion of monovalent cations between different coordination modes was supported by DFT calculations.

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.

Recent Advances in Cancer Imaging with 64CuCl2 PET/CT.

Copper is required for cancer cell proliferation and tumor angiogenesis. Radioactive copper-64 chloride (64CuCl2) is a useful radiotracer for cancer imaging with position emission tomography (PET) based on increased cellular uptake of copper mediated by human copper transporter 1 (hCtr1) expressed on cancer cell membrane. Significant progress has been made in research of using 64CuCl2 as a radiotracer for cancer imaging with PET. Radiation dosimetry study in humans demonstrated radiation safety of 64CuCl2. Recently, 64CuCl2 was successfully used for PET imaging of prostate cancer, bladder cancer, glioblastoma multiforme (GBM), and non-small cell lung carcinoma in humans. Based on the findings from the preclinical research studies, 64CuCl2 PET/CT also holds potential for diagnostic imaging of human hepatocellular carcinoma (HCC), malignant melanoma, and detection of intracranial metastasis of copper-avid tumors based on low physiological background of radioactive copper uptake in the brain. Copper-64 radionuclide emits both β+ and β- particles, suggesting therapeutic potential of 64CuCl2 for radionuclide cancer therapy of copper-avid tumors. Recent progress in production of therapeutic copper-67 radionuclide invites clinical research in use of theranostic pair of 64CuCl2 and 67CuCl2 for cancer imaging and radionuclide therapy.

Reduction in Copper Uptake and Inhibition of Prostate Cancer Cell Proliferation by Novel Steroid-based Compounds.

Knockdown of human copper transporter 1 has been associated with reduction in copper uptake and suppression of prostate cancer cell proliferation and tumor growth. This study evaluated the effects of steroid-based compounds on copper uptake and proliferation of prostate cancer cells based on their anticancer activity and previous docking analysis of steroid-based copper transporter 1 inhibitors. We synthesized several new steroid-based compounds and used 64Cu uptake assay and copper quantification assay with inductively coupled plasma mass spectrometry to study their effects on the cellular copper uptake by prostate cancer cells. Additionally, we used CCK-8 cell proliferation assay to study their effects on the proliferation of prostate cancer cells. Significant reduction in cellular copper uptake was observed in the prostate cancer cells treated with these new steroid-based compounds. Moreover, proliferation of prostate cancer cells was suppressed by treatment with the steroid-based compound 6, which had the strongest copper uptake inhibition activity. Reduction in copper uptake and inhibition of cell proliferation were demonstrated in prostate cancer cells treated with the new steroid-based compounds synthesized in this study. Steroid-based copper transporter 1 inhibitors may become novel anticancer drugs for targeted anti-copper therapy of prostate cancer and other copper hypermetabolic cancers.

At sixes and sevens: cryptic domain in the metal binding chain of the human copper transporter ATP7A

ATP7A and ATP7B are structurally similar but functionally distinct active copper transporters that regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. Both proteins have a chain of six cytosolic metal-binding domains (MBDs) believed to be involved in the copper-dependent regulation of the activity and intracellular localization of these enzymes. Although all the MBDs are quite similar in structure, their spacing differs markedly between ATP7A and ATP7B. We show by NMR that the long polypeptide between MBD1 and MBD2 of ATP7A forms an additional seventh metastable domain, which we called HMA1A (heavy metal associated domain 1A). The structure of HMA1A resembles the MBDs but contains no copper-binding site. The HMA1A domain, which is unique to ATP7A, may modulate regulatory interactions between MBD1–3, contributing to the distinct functional properties of ATP7A and ATP7B.

Nerve Growth Factor Peptides Bind Copper(II) with High Affinity: A Thermodynamic Approach to Unveil Overlooked Neurotrophin Roles.

Nerve growth factor (NGF) is a protein essential to neurons survival, which interacts with its receptor as a non-covalent dimer. Peptides belonging to NGF N-terminal domain are able to mimic the activity of the whole protein. Such activity is affected by the presence of copper ions. The metal is released in the synaptic cleft where proteins, not yet identified, may bind and transfer to human copper transporter 1 (hCtr1), for copper uptake in neurons. The measurements of the stability constants of copper complexes formed by amyloid beta and hCtr1 peptide fragments suggest that beta-amyloid (Aβ) can perform this task. In this work, the stability constant values of copper complex species formed with the dimeric form of N-terminal domain, sequence 1–15 of the protein, were determined by means of potentiometric measurements. At physiological pH, NGF peptides bind one equivalent of copper ion with higher affinity of Aβ and lower than hCtr1 peptide fragments. Therefore, in the synaptic cleft, NGF may act as a potential copper chelating molecule, ionophore or chaperone for hCtr1 for metal uptake. Copper dyshomeostasis and mild acidic environment may modify the balance between metal, NGF, and Aβ, with consequences on the metal cellular uptake and therefore be among causes of the Alzheimer’s disease onset.

Overcoming Radiation Resistance by Iron-Platinum Metal Alloy Nanoparticles in Human Copper Transport 1-Overexpressing Cancer Cells via Mitochondrial Disturbance.

BackgroundRadiation therapy remains an important treatment modality in cancer therapy, however, resistance is a major problem for treatment failure. Elevated expression of glutathione is known to associate with radiation resistance. We used glutathione overexpressing small cell lung cancer cell lines, SR3A-13 and SR3A-14, established by transfection with γ-glutamylcysteine synthetase (γ-GCS) cDNA, as a model for investigating strategies of overcoming radiation resistance. These radiation-resistant cells exhibit upregulated human copper transporter 1 (hCtr1), which also transports cisplatin. This study was initiated to investigate the effect and the underlying mechanism of iron-platinum nanoparticles (FePt NPs) on radiation sensitization in cancer cells.Materials and MethodsUptakes of FePt NPs in these cells were studied by plasma optical emission spectrometry and transmission electron microscopy. Effects of the combination of FePt NPs and ionizing radiation were investigated by colony formation assay and animal experiment. Intracellular reactive oxygen species (ROS) were assessed by using fluorescent probes and imaged by a fluorescence-activated-cell-sorting caliber flow cytometer. Oxygen consumption rate (OCR) in mitochondria after FePt NP and IR treatment was investigated by a Seahorse XF24 cell energy metabolism analyzer.ResultsThese hCtr1-overexpressing cells exhibited elevated resistance to IR and the resistance could be overcome by FePt NPs via enhanced uptake of FePt NPs. Overexpression of hCtr1 was responsible for the increased uptake/transport of FePt NPs as demonstrated by using hCtr1-transfected parental SR3A (SR3A-hCtr1-WT) cells. Increased ROS and drastic mitochondrial damages with substantial reduction of oxygen consumption rate were observed in FePt NPs and IR-treated cells, indicating that structural and functional insults of mitochondria are the lethal mechanism of FePt NPs. Furthermore, FePt NPs also increased the efficacy of radiotherapy in mice bearing SR3A-hCtr1-WT-xenograft tumors.ConclusionThese results suggest that FePt NPs can potentially be a novel strategy to improve radiotherapeutic efficacy in hCtr1-overexpressing cancer cells via enhanced uptake and mitochondria targeting.

Exploration of the Potential Role for Aβ in Delivery of Extracellular Copper to Ctr1.

Amyloid beta (Aβ) peptides are notorious for their involvement in Alzheimer's disease (AD), by virtue of their propensity to aggregate to form oligomers, fibrils, and eventually plaques in the brain. Nevertheless, they appear to be essential for correct neurophysiology on the synaptic level and may have additional functions including antimicrobial activity, sealing the blood-brain barrier, promotion of recovery from brain injury, and even tumor suppression. Aβ peptides are also avid copper chelators, and coincidentally copper is significantly dysregulated in the AD brain. Copper (Cu) is released in significant amounts during calcium signaling at the synaptic membrane. Aβ peptides may have a role in maintaining synaptic Cu homeostasis, including as a scavenger for redox-active Cu and as a chaperone for clearing Cu from the synaptic cleft. Here, we employed the Aβ1-16 and Aβ4-16 peptides as well-established non-aggregating models of major Aβ species in healthy and AD brains, and the Ctr1-14 peptide as a model for the extracellular domain of the human cellular copper transporter protein (Ctr1). With these model peptides and a number of spectroscopic techniques, we investigated whether the Cu complexes of Aβ peptides could provide Ctr1 with either Cu(II) or Cu(I). We found that Aβ1-16 fully and rapidly delivered Cu(II) to Ctr1-14 along the affinity gradient. Such delivery was only partial for the Aβ4-16/Ctr1-14 pair, in agreement with the higher complex stability for the former peptide. Moreover, the reaction was very slow and took ca. 40 h to reach equilibrium under the given experimental conditions. In either case of Cu(II) exchange, no intermediate (ternary) species were present in detectable amounts. In contrast, both Aβ species released Cu(I) to Ctr1-14 rapidly and in a quantitative fashion, but ternary intermediate species were detected in the analysis of XAS data. The results presented here are the first direct evidence of a Cu(I) and Cu(II) transfer between the human Ctr1 and Aβ model peptides. These results are discussed in terms of the fundamental difference between the peptides' Cu(II) complexes (pleiotropic ensemble of open structures of Aβ1-16 vs the rigid closed-ring system of amino-terminal Cu/Ni binding Aβ4-16) and the similarity of their Cu(I) complexes (both anchored at the tandem His13/His14, bis-His motif). These results indicate that Cu(I) may be more feasible than Cu(II) as the cargo for copper clearance from the synaptic cleft by Aβ peptides and its delivery to Ctr1. The arguments in favor of Cu(I) include the fact that cellular Cu export and uptake proteins (ATPase7A/B and Ctr1, respectively) specifically transport Cu(I), the abundance of extracellular ascorbate reducing agent in the brain, and evidence of a potential associative (hand-off) mechanism of Cu(I) transfer that may mirror the mechanisms of intracellular Cu chaperone proteins.

Nanobodies against the metal binding domains of ATP7B as tools to study copper transport in the cell.

Nanobodies are genetically engineered single domain antibodies derived from the unusual heavy-chain only antibodies found in llamas and camels. The small size of the nanobodies and flexible selection schemes make them uniquely versatile tools for protein biochemistry and cell biology. We have developed a panel of nanobodies against the metal binding domains of the human copper transporter ATP7B, a multidomain membrane protein with a complex regulation of enzymatic activity and intracellular localization. To enable the use of the nanobodies as tools to investigate copper transport in the cell, we characterized their binding sites and affinity by isothermal titration calorimetry and NMR. We have identified nanobodies against each of the first four metal binding domains of ATP7B, with a wide affinity range, as evidenced by dissociation constants from below 10-9 to 10-6 M. We found both the inhibitory and activating nanobodies among those tested. The diverse properties of the nanobodies make the panel useful for the structural studies of ATP7B, immunoaffinity purification of the protein, modulation of its activity in the cell, protein dynamics studies, and as mimics of copper chaperone ATOX1, the natural interaction partner of ATP7B.

Preclinical PET imaging study of lung cancer with 64CuCl2.

Human copper transporter 1 (CTR1) has been proven to be overexpressed in many types of cancer cells, and copper (II)-64 chloride (64CuCl2) has been used as an effective tracer for positron emission tomography (PET) imaging in tumor-bearing animal models. Thus, this study aimed to investigate the potential application of 64CuCl2 in PET imaging of lung cancer through targeting CTR1. The expression of CTR1 in a series of lung cancer cell lines was identified by quantitative real-time polymerase chain reaction (Q-PCR), western blot, enzyme-linked immunosorbnent assay (ELISA), and immunofluorescent staining. Then in vitro cell uptake assay of 64CuCl2 was investigated in human lung cancer cell lines with different levels of CTR1 expression. Small animal PET imaging and quantitative analysis were performed in human lung cancer tumor-bearing mice after intravenous injection of 64CuCl2, respectively. The CTR1 expression in multiple human lung cancer cells was identified and confirmed, and H1299 cell lines with high CTR1 expression, H460 with moderate CTR1, and H1703 with low CTR1 were selected for further experiments. In vitro cellular uptake assay displayed that the 64CuCl2 uptake by these three kinds of cells was positively correlated with their CTR1 expressed levels. The blocking experiments testified the specificity of 64CuCl2 to target CTR1. Moreover, small animal PET imaging and quantitative results showed that 64CuCl2 accumulation in H1299, H460, and H1703 tumor-bearing mice were consistent with CTR1 levels and cell uptake experiments. The expression of CTR1 in human lung cancer xenograft model could be successfully visualized by 64CuCl2 PET examination. With the expected growth of PET/CT examination to be an essential strategy in clinical lung cancer management, 64CuCl2 has the potential to be a promising PET imaging agent of lung cancer.

Cu(I) Controls Conformational States in Human Atox1 Metallochaperone: An EPR and Multiscale Simulation Study.

Atox1 is a human copper metallochaperone that is responsible for transferring copper ions from the main human copper transporter, hCtr1, to ATP7A/B in the Golgi apparatus. Atox1 interacts with the Ctr1 C-terminal domain as a dimer, although it transfers the copper ions to ATP7A/B in a monomeric form. The copper binding site in the Atox1 dimer involves Cys12 and Cys15, while Lys60 was also suggested to play a role in the copper binding. We recently showed that Atox1 can adopt various conformational states, depending on the interacting protein. In the current study, we apply EPR experiments together with hybrid quantum mechanics–molecular mechanics molecular dynamics simulations using a recently developed semiempirical density functional theory approach, to better understand the effect of Atox1’s conformational states on copper coordination. We propose that the flexibility of Atox1 occurs owing to protonation of one or more of the cysteine residues, and that Cys15 is an important residue for Atox1 dimerization, while Cys12 is a critical residue for Cu(I) binding. We also show that Lys60 electrostatically stabilizes the Cu(I)–Atox1 dimer.

A new metal affinity NCTR25 tag as a better alternative to the His-tag for the expression of recombinant fused proteins

His-tagging is commonly used in fusion protein production, but the His-tag is usually prohibited in medicinal proteins and must be removed. A fragment (NCTR25-tag) truncated from the N-terminus of human copper transporter 1 was tested for feasibility as a replacement for the His-tag in fusion proteins. The NCTR25-tag and His-tag were separately fused to the transthyretin (TTR) protein, and the expression, affinity purification, refolding and stability of the two kinds of fusions were compared. NCTR25 fusion produced a 63% higher yield of the recombinant protein, which was purified by metal affinity chromatography with an efficiency similar to that of His-tagged protein. NCTR25-tag fusion had much less impact on the foldability, kinetic and thermodynamic stability of tetrameric TTR than His-tag fusion. When the tags were individually fused to enhanced green fluorescent protein (EGFP), NCTR25 fusion yielded 29–128% more product than His-EGFP. NCTR25-EGFP could be purified by metal affinity chromatography and showed better foldability than His-EGFP. Furthermore, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) fusion with the third disulfide loop of TGF-α (TGF3L-TRAIL) fused with the NCTR25-tag retained the stability and superactivity of His-TGF3L-TRAIL. Therefore, the native tag NCTR25-tag is a feasible alternative to the His-tag in medicinal recombinant proteins.

Cu(II) Binding to the N-Terminal Model Peptide of the Human Ctr2 Transporter at Lysosomal and Extracellular pH.

It was shown that His3 of human copper transporter 1 (hCtr1) prompts the ATCUN-like Cu(II) coordination for model peptides of the hCtr1 N-terminus. Its high Cu(II) affinity is a potential driving force for the transfer of Cu(II) from extracellular Cu(II) carriers to hCtr1. Having a sequence similar to that of hCtr1, hCtr2 has been proposed as another human copper transporter. However, the N-terminal domain of hCtr2 is much shorter than that of hCtr1, with different copper binding motifs at its N-terminus. Employing a model peptide of the hCtr2 N-terminus, MAMHF-am, we demonstrated that His4 provides a unique pattern of Cu(II) complexes, involving Met sulfurs in their Cu(II) coordination sphere. The affinity of Cu(II) for MAMHF-am is a few orders of magnitude lower than that reported for the hCtr1 model peptides at the extracellular pH of 7.4, suggesting a maximal complementary role of Cu(II) binding to hCtr2 in the import of copper from the extracellular space to the cytoplasm. On the other hand, the ability of the hCtr2 model peptide to capture Cu(II) from amino acids and short peptides (potential degradation products of proteins) at pH 5.0 and the known predominant lysosomal localization of hCtr2 support an important potential role of the Cu(II)-hCtr2 interaction in the recovery of copper from lysosomes.