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

Shadi Maghool,Sharon La Fontaine,Blaine R. Roberts,Megan J. Maher,Ann H. Kwan

doi:10.1038/s41598-020-60953-z

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 P1B-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.

Highlights

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
This study proposed a model for Cu binding and transport by ATP7B, where the metal binding domains (MBDs) 4 receives Cu from Atox[1] and passes the metal to MBD 6, which transfers Cu to MBD 5 for delivery to the transmembrane domain (TMD) for translocation across the membrane[21]
Purified apo-proteins were loaded with Cu(I) as previously described[10] and after size exclusion chromatography (SEC) to remove excess Cu(I), were analyzed for Cu(I) content

Summary

Introduction

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. ATP7A (Menkes disease protein, MNK) and ATP7B (Wilson disease protein, WLN) are two Cu(I)-specific P1B-type ATPases that are essential for Cu transport and homeostasis These ion pumps, which www.nature.com/scientificreports are localized in the trans-Golgi network (TGN), utilize ATP hydrolysis to transport the metal across the TGN membrane to activate Cu-dependent enzymes within the secretory pathway A distinguishing feature of the ATP7A and ATP7B proteins is the presence of a large amino-terminal extension that is composed of six Cu-binding domains (~600 residues in total) These metal binding domains (MBDs) have ferredoxin-like βαββαβ folds and bind Cu(I) at C-XX-C motifs, which redox cycle between oxidized (disulfide) and reduced (thiol) states[7,24,25,26,27,28,29,30,31,32,33]. The MBDs participate in Cu exchange via protein-protein interactions, which are facilitated by flexible polypeptide linkers that bridge the domains[34,35]

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