Human glutaredoxin-1 can transfer copper to isolated metal binding domains of the P1B-type ATPase, ATP7B

Abstract

Intracellular copper (Cu) in eukaryotic organisms is regulated by homeostatic systems, which rely on the activities of soluble metallochaperones that participate in Cu exchange through highly tuned protein-protein interactions. Recently, the human enzyme glutaredoxin-1 (hGrx1) has been shown to possess Cu metallochaperone activity. The aim of this study was to ascertain whether hGrx1 can act in Cu delivery to the metal binding domains (MBDs) of the P 1B-type ATPase ATP7B and to determine the thermodynamic factors that underpin this activity. hGrx1 can transfer Cu to the metallochaperone Atox1 and to the MBDs 5-6 of ATP7B (WLN5-6). This exchange is irreversible. In a mixture of the three proteins, Cu is delivered to the WLN5-6 preferentially, despite the presence of Atox1. This preferential Cu exchange appears to be driven by both the thermodynamics of the interactions between the proteins pairs and of the proteins with Cu(I). Crucially, protein-protein interactions between hGrx1, Atox1 and WLN5-6 were detected by NMR spectroscopy both in the presence and absence of Cu at a common interface. This study augments the possible activities of hGrx1 in intracellular Cu homeostasis and suggests a potential redundancy in this system, where hGrx1 has the potential to act under cellular conditions where the activity of Atox1 in Cu regulation is attenuated. Copper (Cu) is a redox active metal and an essential trace element for human health 1,2. In biological systems, Cu is found in two oxidation states, reduced Cu(I) (cuprous) and oxidized Cu(II) (cupric). These reversible states, which allow Cu to serve as both an oxidant and reductant, render it an exceptional cofactor for redox enzymes known as oxidoreductases 1,2 , however excess intracellular concentrations of Cu are toxic and lead to cellular oxidative damage 3,4. In particular, the reaction of hydrogen peroxide with Cu(I) and the reduction of Cu(II) by superoxide, produce hydroxyl radicals that damage proteins, nucleic acids and lipids 5,6. In humans, the dysregulation of Cu metabolism is associated with diseases such as Menkes syndrome, Wilson's disease, prion diseases, Alzheimer's disease and fatal motor neuron diseases such as familial amyotrophic lateral sclerosis 7-12. To maintain the fine balance between cellular Cu requirements and toxicity, organisms have evolved sophisticated metalloregulatory systems for the coordination of metals such as Cu to proteins within the cell, and the controlled transfer of these metals between protein partners. Soluble proteins that are responsible for Cu binding and transfer are termed 'metallochaperones' 3 , which transport Cu in its reduced state (Cu(I)), often via coordination with cysteine residues 13,14. In eukaryotes, including humans, Cu concentrations are regulated through control of the levels of the Cu import protein, copper transporter 1 (Ctr1) 15,16 at the plasma membrane and the trafficking and/or activity of the Cu export proteins, ATP7A or ATP7B 17-19. In humans, ATP7A (Menkes disease protein, MNK) and ATP7B (Wilso...

The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb3-Type Cytochrome Oxidase Biogenesis

“…While the inactivation of the glutaredoxin-like N-MBS1 can be fully restored by Cu supplementation, the CopZ-like N-MBS2 yielded an intermediate phenotype that could only be rescued partially by Cu addition. Considering that the CopZ-like N-MBS2 is conserved between CcoI and CopA, while the presence of the N-MBS1 is a particular feature of the high-affinity CcoI, these findings suggested that N-MBS1 might be important for receiving Cu from an alternative Cu donor different than CopZ, such as glutaredoxin or even glutathione ( Maghool et al, 2020 ; Stewart et al, 2020 ).…”

“…While the TM-MBSs are essential for Cu translocation ( Gonzalez-Guerrero et al, 2008 , 2009 ), the N-MBSs in bacterial CopA-like ATPases appear to be non-essential ( Fan et al, 2001 ; Gonzalez-Guerrero and Arguello, 2008 ; Drees et al, 2015 ). Instead, they are proposed to regulate the ATPase activity in response to Cu availability ( Wu et al, 2008 ; Arguello et al, 2016 ) and to support Cu-loading of the TM-MBSs ( Figure 7 ; Drees et al, 2015 ; Maghool et al, 2020 ). Current models suggest that in the absence of Cu, the N-MBS is interacting with the N- and the A-domains of CopA, preventing either ATP binding or phosphorylation of the P-domain ( Tsivkovskii et al, 2001 ; Wu et al, 2008 ; Gonzalez-Guerrero et al, 2009 ).…”

“…The absence of the Met residue in the N-MBS2 of CcoI could potentially position the cysteine residues in a more flexible conformation, suitable to receive Cu from alternative Cu donors, including glutaredoxin and small molecules, like glutathione or cysteine ( Figure 7 ; Helbig et al, 2008 ; Banci et al, 2010c ; Singleton et al, 2010 ; Aliaga et al, 2016 ; Bhattacharjee et al, 2017 ; Dolgova et al, 2017 ). These molecules have been linked to Cu metabolism in other species ( Li et al, 2004 ; Helbig et al, 2008 ) and have been shown to interact with P 1B -type ATPases ( Rosenzweig et al, 1999 ; Maghool et al, 2020 ). A CopZ independent Cu transfer to CcoI would also explain, why cbb 3 -Cox activity is reduced by only 30% in the absence of CopZ ( Utz et al, 2019 ).…”

Silver Binding to Bacterial Glutaredoxins Observed by NMR

“…One hypothesis for this specificity that had been proposed is that the proline in the GRX active site prevents metal binding and/or the dimerization required for metal transfer [14]. However, more recent research has indicated that copper ions can bind within GRX's active site unhindered by these prolines [15,16]. Instead, other metallochaperones, such as ATOX1, with a higher metal affinity, outcompete GRX for metal cations [15].…”

The Caenorhabditis elegans homolog of human copper chaperone Atox1, CUC-1, aids in distal tip cell migration

“…In human cells, after copper transporter protein 1 (Ctr1)-mediated uptake, the cytoplasmic Cu chaperone Atox1 transports Cu to ATP7A and ATP7B in the trans-Golgi network (Puig and Thiele 2002). In support of possible redundancy, it was recently shown that glutaredoxin 1 could replace Atox1 and deliver Cu to ATP7B (Maghool et al 2020). ATP7A/B are P 1B -type ATPases that use ATP hydrolysis to transfer Cu to the lumen for loading of target Cu-dependent enzymes, such as ceruloplasmin, tyrosinase and lysyl oxidase (Matson Dzebo et al 2016).…”