Interactions between Copper-binding Sites Determine the Redox Status and Conformation of the Regulatory N-terminal Domain of ATP7B

Erik S Leshane,Ujwal Shinde,Joel M Walker,Amanda N Barry,Ninian J Blackburn,Martina Ralle,Svetlana Lutsenko

doi:10.1074/jbc.m109.074633

Abstract

Copper-transporting ATPase ATP7B is essential for human copper homeostasis and normal liver function. ATP7B has six N-terminal metal-binding domains (MBDs) that sense cytosolic copper levels and regulate ATP7B. The mechanism of copper sensing and signal integration from multiple MBDs is poorly understood. We show that MBDs communicate and that this communication determines the oxidation state and conformation of the entire N-terminal domain of ATP7B (N-ATP7B). Mutations of copper-coordinating Cys to Ala in any MBD (2, 3, 4, or 6) change the N-ATP7B conformation and have distinct functional consequences. Mutating MBD2 or MBD3 causes Cys oxidation in other MBDs and loss of copper binding. In contrast, mutation of MBD4 and MBD6 does not alter the redox status and function of other sites. Our results suggest that MBD2 and MBD3 work together to regulate access to other metal-binding sites, whereas MBD4 and MBD6 receive copper independently, downstream of MBD2 and MBD3. Unlike Ala substitutions, the Cys-to-Ser mutation in MBD2 preserves the conformation and reduced state of N-ATP7B, suggesting that hydrogen bonds contribute to interdomain communications. Tight coupling between MBDs suggests a mechanism by which small changes in individual sites (induced by copper binding or mutation) result in stabilization of distinct conformations of the entire N-ATP7B and altered exposure of sites for interactions with regulatory proteins.

Highlights

To better understand the relationship between metal-binding subdomains (MBDs) in the protein and elution of Atox1 were accomplished by incubating context of a fully folded N-terminal domain of ATP7B (N-ATP7B), we characterized the effect resin-bound fusion protein with buffer containing 50 mM dithioof mutating individual sites on copper binding by N-ATP7B. threitol, 25 mM NaH2PO4, and 150 mM NaCl for 36 h at Our studies revealed the key roles of MBD2 and MBD3 in room temperature
MBD2 Influences the Copper-binding Capability of Other N-terminal MBDs—To understand the relationships between different MBDs, we first generated and characterized the m2A mutant of N-ATP7B, in which the copper-binding CxxC motif of MBD2 was replaced with AxxA (Fig. 2A)
We hypothesized that MBD2 performs this gating role by interacting with other MBDs and changing the protein conformation of N-ATP7B in response to copper binding

Summary

Introduction

N-ATP7B is phosphorylated by a kinase in response to copper binding (3), houses the sequence determinants for the copper-dependent apical targeting of ATP7B (4), and recognizes dynactin, a component of the trafficking machinery, in a copper-dependent manner (5). It remains unknown whether all of these copper-induced events are directly coupled. The three-dimensional structure of N-ATP7B is unknown; the structures of individual subdomains and two pairs (MBD3/4 and MBD5/6) have been solved (8 –15) These studies revealed that all MBDs have a ferridoxin-like ␤␣␤␤␣␤-fold, with the coppercoordinating site situated in a solvent-exposed loop between the ␤1-strand and ␣1-helix (Fig. 1B). Cross-talk between Metal-binding Sites in ATP7B for small perturbations in structure (because of mutation or copper binding) to alter the tertiary structure of the entire N-ATP7B

Methods

Results

Conclusion