ATOX1 Human

What is the primary function of human ATOX1 protein?

Human antioxidant protein 1 (ATOX1) is a small cytosolic protein of 68 amino acid residues that plays an essential role in copper homeostasis. It functions primarily as a copper chaperone, binding and shuttling copper from the cellular copper transporter (Ctr1) to copper-transporting ATPases ATP7A and ATP7B located in the trans-Golgi network and various endocytic vesicles . This copper transfer is crucial for the maturation of copper-dependent enzymes within the secretory pathway and for maintaining appropriate copper levels in the cytosol and mitochondria .

The copper transport function of ATOX1 enables activation of numerous copper-dependent enzymes involved in:

• Neurotransmitter biosynthesis

• Iron efflux

• Neovascularization

• Wound healing

• Regulation of blood pressure

Beyond copper transport, ATOX1 demonstrates antioxidant properties, protecting cells against hydrogen peroxide-induced oxidative damage and other oxidative stressors . It was originally identified in Saccharomyces cerevisiae as a molecule capable of antioxidant defense, hence its name (Atx1 in yeast) . Recent research has revealed additional cellular roles for ATOX1, including modulation of cancer therapy responses, contribution to inflammatory responses, and protection against various oxidative stresses .

What is the structure of human ATOX1 and how does it bind copper?

ATOX1 is a small protein with 68 amino acid residues that is highly conserved across different phyla, showing 85% sequence identity among mammalian species . The protein contains three cysteine residues, with Cys12 and Cys15 forming the primary copper-binding site . The third cysteine (Cys41) does not participate in copper binding and is accessible for experimental modifications such as spin-labeling .

ATOX1 can adopt different conformational states:

• A closed conformation consistent with the crystal structure (PDB ID: 1fee)

• An open conformation that is more flexible and facilitates interactions with various parts of the hCtr1 intracellular domains

Copper coordination in ATOX1 fluctuates between three- and four-coordinated states in the closed conformation . QM/MM molecular dynamics simulations have revealed that:

• Cu-Cys12 coordination has a monomodal distribution with average distances of 2.15 ± 0.01 Å

• Cu-Cys15 coordination shows a bimodal distribution with average distances of 2.8 ± 0.1 Å, resulting from one of the two Cys15 residues flipping between first and second shell coordination

• Lys60 electrostatically stabilizes the Cu(I)-ATOX1 dimer and shows a bimodal distance distribution with Cys15

ATOX1 interacts with the Ctr1 C-terminal domain as a dimer, although it transfers copper ions to ATP7A/B in a monomeric form . This structural flexibility is crucial for ATOX1's function in copper transport and its interactions with different protein partners.

How does ATOX1 interact with other proteins in the copper transport pathway?

ATOX1 serves as a critical intermediary in the copper transport pathway, interacting with multiple proteins to facilitate copper movement throughout the cell. At the plasma membrane, ATOX1 receives copper from the human copper transporter hCtr1, interacting with hCtr1's C-terminal domain as a dimer . This interaction is facilitated by ATOX1's ability to adopt an open conformation that is more flexible and can interact with various parts of the hCtr1 intracellular domains .

After binding copper, ATOX1 transfers it to the copper-transporting ATPases ATP7A and ATP7B located in the trans-Golgi network and various endocytic vesicles . This transfer occurs with ATOX1 in a monomeric form . The interaction involves direct binding between ATOX1 and the N-terminal copper binding domain (NBD) of ATP7A/B, with copper transfer occurring to various metal binding sites in NBD .

The functional significance of these interactions is demonstrated by the fact that ATP7B's ATPase activity is stimulated specifically by Cu-ATOX1 (copper-bound ATOX1) but not by apo-ATOX1 (ATOX1 without copper) . Studies of bacterial copper transporting ATPases have revealed that in the absence of NBD, the intramembrane copper transport site can still receive metal from the chaperone, suggesting potential alternative mechanisms for copper transfer .

Beyond the copper transport pathway, ATOX1 interacts with several subunits of the anaphase-promoting complex (APC), which is involved in cell cycle regulation, particularly in facilitating sister chromatid separation during mitosis . These interactions suggest that ATOX1 plays roles beyond copper transport, potentially linking copper homeostasis with cell cycle progression.

How do the conformational states of ATOX1 influence its copper coordination and function?

ATOX1 exhibits remarkable structural flexibility that directly impacts its copper coordination and functional capabilities. Research has identified two primary conformational states: a closed conformation consistent with the crystal structure and an open conformation that demonstrates greater flexibility . These states are not static but dynamically interconvert depending on protein interactions and environmental conditions.

In the closed conformation, QM/MM molecular dynamics simulations have revealed that copper coordination fluctuates between three- and four-coordinated states . This fluctuation arises from the behavior of the cysteine residues, particularly Cys15, which can flip between first and second shell coordination. Specifically:

• Cu-Cys12 coordination maintains a consistent monomodal distribution with average distances of 2.15 ± 0.01 Å

• Cu-Cys15 coordination demonstrates a bimodal distribution with average distances of 2.8 ± 0.1 Å

• Lys60-Cys15 distance distribution is also bimodal, reflecting the same flipping behavior of Cys15

The flexibility of ATOX1 appears to be regulated by the protonation state of its cysteine residues, with experimental evidence suggesting that Cys15 is crucial for ATOX1 dimerization, while Cys12 is critical for Cu(I) binding . Additionally, Lys60 plays an important role in electrostatically stabilizing the Cu(I)-ATOX1 dimer .

These conformational dynamics are functionally significant: the open conformation facilitates ATOX1's interaction with hCtr1 for receiving copper, while transitions to other conformational states may be necessary for interaction with ATP7A/B and other protein partners . The ability to adopt different conformations depending on the interacting protein represents a sophisticated mechanism for regulating copper transfer and potentially integrating this process with other cellular functions.

What mechanisms explain ATOX1's dual roles in copper transport and antioxidant defense?

ATOX1 was originally identified as an antioxidant protein in yeast (Atx1) before its copper chaperone function was established in mammals . This dual functionality raises important questions about whether these roles are mechanistically linked or represent distinct activities. Current evidence suggests several potential mechanisms that may explain this dual functionality:

• Redox-sensitive copper binding site: The copper-binding site of ATOX1, involving Cys12 and Cys15, is sensitive to the cytosolic redox environment . This sensitivity creates a direct link between copper binding/transport and cellular redox status, potentially enabling ATOX1 to adjust its activity based on oxidative conditions.

• Copper sequestration as antioxidant defense: By binding copper ions, ATOX1 prevents them from participating in Fenton reactions that generate harmful reactive oxygen species. This sequestration function represents a passive antioxidant mechanism that is directly linked to its copper chaperone role.

• Conformational state switching: ATOX1's ability to adopt different conformational states depending on interacting proteins and environmental conditions may allow it to switch between copper transport and antioxidant functions . The flexibility of ATOX1 occurs due to protonation of one or more cysteine residues, directly linking conformation to redox conditions .

• Redox signaling through protein interactions: ATOX1 interactions with non-copper transport proteins, such as APC subunits , may enable it to influence cellular processes in response to redox changes, potentially explaining how it contributes to inflammatory responses .

What is the role of ATOX1 in cell cycle regulation and cancer biology?

Recent research has uncovered unexpected roles for ATOX1 in cell cycle regulation and cancer biology that extend beyond its classical function in copper transport. ATOX1 interacts with several subunits of the anaphase-promoting complex (APC), a crucial regulator of cell cycle progression that facilitates the separation of sister chromatids during mitosis . This interaction has direct functional consequences, as cells lacking ATOX1 exhibit a prolonged G phase in the cell cycle .

The APC is considered a potential target for anticancer agents due to its central role in cell cycle regulation . ATOX1's interaction with APC components suggests it may influence cancer cell division through this pathway, potentially explaining observations that changing levels of ATOX1 modulate responses to cancer therapies .

Beyond cell cycle regulation, ATOX1 has been found to localize at lamellipodia edges in breast cancer cells, where it promotes cancer cell migration through mechanisms that remain to be elucidated . This observation connects ATOX1 to cancer metastasis, one of the most clinically challenging aspects of cancer progression.

The link between ATOX1 and cancer may involve multiple mechanisms:

• Direct effects on cell cycle regulation through APC interactions

• Modulation of copper availability for enzymes involved in angiogenesis and metastasis

• Influence on cellular redox status, which can affect cancer cell survival and therapeutic responses

• Potential roles in transcriptional regulation, as suggested by interactions with DNA/RNA-binding proteins

These emerging functions position ATOX1 at the intersection of copper homeostasis, redox regulation, and cell cycle control, making it a potentially valuable target for understanding cancer biology and developing novel therapeutic approaches.

What experimental approaches are optimal for studying ATOX1 copper binding and transfer?

Investigating ATOX1's copper binding and transfer mechanisms requires specialized experimental approaches that can capture the structural dynamics and molecular interactions involved. Based on current research methodologies, optimal approaches include:

Spectroscopic Methods:

• Electron Paramagnetic Resonance (EPR) techniques provide valuable insights into copper coordination states and protein conformational changes:

  • Continuous Wave EPR (CW-EPR) can be performed at room temperature (295 ± 2 K) using specific parameters (microwave power of 20.0 mW, modulation amplitude of 1.0 G)

  • Double Electron-Electron Resonance (DEER) experiments conducted at low temperature (50 ± 1.0 K) on Q-band instruments enable measurement of distances between labeled sites

Protein Labeling Strategies:

• Site-directed spin labeling, particularly at Cys41 (as Cys12 and Cys15 are involved in copper binding), using methanesulfonothioate (MTSSL) enables EPR measurements of conformational changes

• Fluorescent labeling strategies can be employed for real-time monitoring of protein dynamics and interactions

Computational Methods:

• Quantum Mechanics/Molecular Mechanics (QM/MM) molecular dynamics simulations provide atomic-level insights into copper coordination dynamics

• Density Functional Theory (DFT)-based calculations characterize the coordination state of Cu(I)

• Principal Component Analysis (PCA) helps identify functionally relevant collective motions from simulation trajectories

Biochemical and Structural Approaches:

• X-ray crystallography of ATOX1 in different states (apo, copper-bound, in complex with partners)

• NMR spectroscopy for studying protein dynamics and interactions in solution

• ATPase activity assays to monitor functional copper transfer to ATP7A/B

• Co-immunoprecipitation coupled with mass spectrometry to identify interaction partners

The optimal research strategy would integrate multiple techniques to correlate structural changes with functional outcomes. For example, combining spectroscopic measurements of copper coordination with functional assays of copper transfer efficiency would provide a more complete picture of ATOX1's mechanism than either approach alone.

How can researchers effectively differentiate between copper-dependent and copper-independent functions of ATOX1?

Distinguishing between ATOX1's copper-dependent and copper-independent functions represents a significant challenge in understanding this multifunctional protein. Several methodological approaches can be employed to effectively make this distinction:

Mutation-Based Approaches:

• Site-directed mutagenesis of copper-binding residues (Cys12 and Cys15) can selectively disrupt copper coordination while potentially preserving other functional domains

• Differential mutation of Cys12 (critical for Cu(I) binding) versus Cys15 (important for dimerization) can help separate these functions

• Lys60 mutations can disrupt the electrostatic stabilization of Cu(I)-ATOX1 without directly affecting the copper-binding site

Copper Availability Manipulation:

• Comparison of apo-ATOX1 versus Cu-ATOX1 effects on specific cellular processes

• Use of copper chelators to reduce cellular copper availability and observe which ATOX1 functions are affected

• Copper supplementation experiments to determine which phenotypes can be rescued by restoring copper availability

Protein Interaction Analysis:

• Comparative interaction profiling of apo-ATOX1 versus Cu-ATOX1 using proximity ligation assays or co-immunoprecipitation

• Analysis of which protein interactions (e.g., with APC subunits) depend on ATOX1's copper-binding status

Functional Assays with Controlled Variables:

• Cell cycle analysis in the presence of ATOX1 variants with altered copper binding, comparing effects on G phase duration

• Assessment of antioxidant capacity using hydrogen peroxide challenge assays with wild-type versus copper-binding mutants of ATOX1

• Evaluation of transcriptional regulation activities under conditions of copper excess or depletion

Redox State Manipulation:

• Since "the copper-binding site of ATOX1 is sensitive to cytosolic redox environment," manipulating cellular redox conditions can help determine which functions depend on this redox sensitivity

By systematically implementing these approaches and integrating results from multiple experimental systems, researchers can build a comprehensive understanding of which ATOX1 functions require its copper-binding capability and which operate independently of copper.

What spectroscopic methods are most suitable for analyzing ATOX1 conformational changes?

ATOX1 undergoes significant conformational changes during its functional cycle, transitioning between open and closed states as it interacts with different protein partners. Capturing these dynamic structural changes requires specialized spectroscopic approaches:

Electron Paramagnetic Resonance (EPR) Spectroscopy:

• Continuous Wave EPR (CW-EPR) can be performed using an X-band spectrometer (9.0-9.5 GHz) at room temperature with specific parameters (microwave power of 20.0 mW, modulation amplitude of 1.0 G)

• Data analysis can be conducted using computational tools such as the easyspin toolbox in MATLAB

• This technique requires spin-labeling of accessible cysteine residues, with Cys41 being the primary target for ATOX1 since Cys12 and Cys15 are involved in copper binding

Double Electron-Electron Resonance (DEER):

• The constant time four-pulse DEER experiment provides distance measurements between labeled sites

• Optimal parameters include conducting measurements at 50 ± 1.0 K on a Q-band instrument with specific pulse settings

• Data analysis can be performed using programs such as DeerAnalysis with Tikhonov regularization

• This approach provides valuable information about the distribution of conformational states

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• NMR has been used to study ATOX1 structure in solution, including monomeric forms

• This technique is particularly valuable for monitoring dynamic changes in protein structure under various conditions

• Chemical shift perturbation experiments can map interaction surfaces with partner proteins

X-ray Crystallography:

• While not capturing dynamics directly, comparing crystal structures of ATOX1 in different states provides structural snapshots that complement dynamic studies

• The crystal structure (PDB ID: 1fee) has served as an important reference point for computational studies

Fluorescence-Based Techniques:

• Förster Resonance Energy Transfer (FRET) with strategically placed fluorophores can monitor conformational changes in real-time

• Single-molecule FRET is particularly valuable for capturing the distribution of conformational states

For optimal results, researchers should combine multiple spectroscopic approaches, correlating the structural information with computational analyses such as Principal Component Analysis (PCA) to distinguish functionally relevant motions from random thermal fluctuations . Interpretation of spectroscopic data benefits significantly from computational simulations that can provide atomic-level insights into the observed conformational changes.

What explains the emerging diversity of ATOX1 interactions with non-copper transport proteins?

The discovery that ATOX1 interacts with proteins beyond the copper transport pathway, particularly cell cycle regulators like the anaphase-promoting complex (APC) , raises important questions about the functional significance and evolution of these diverse interactions. Several mechanisms may explain this expanding interaction network:

Functional Integration Hypothesis:
ATOX1 may serve as a molecular link between copper homeostasis and other cellular processes, allowing coordination of copper utilization with cellular demands that change during processes like cell division, differentiation, or stress response. This integration would enable cells to adapt copper distribution based on physiological needs or environmental challenges.

Conditional Interaction Model:
ATOX1's conformational flexibility allows it to adopt different states depending on copper binding, redox conditions, and partner proteins . These conformational changes likely expose different interaction surfaces, enabling ATOX1 to form context-specific protein complexes. The open conformation identified in research is more flexible and can interact with various parts of the hCtr1 intracellular domains , suggesting similar flexibility may facilitate other protein interactions.

Copper-Sensing Regulatory Mechanism:
ATOX1's copper-binding status may influence its interactions with non-copper transport proteins, serving as a mechanism to regulate their activity based on copper availability. This would effectively make ATOX1 a copper sensor that transmits information about copper status to multiple cellular pathways.

Evolutionary Repurposing:
The diversity of ATOX1 interactions may reflect evolutionary adaptation, where a protein originally specialized for copper transport has been repurposed to serve additional functions. This evolutionary history is suggested by ATOX1's high conservation (85% sequence identity in mammalian species) while acquiring species-specific functions.

Cellular Stress Response Coordination:
Given ATOX1's antioxidant properties and ability to protect cells against oxidative stresses , its interactions with non-copper transport proteins might represent mechanisms to coordinate responses to different types of cellular stress, including oxidative damage, copper imbalance, and cell cycle dysregulation.