The Different Intermolecular Interactions of the Soluble Copper-binding Domains of the Menkes Protein, ATP7A

Lucia Banci,Ivano Bertini,Francesca Cantini,Nunzia Della-Malva,Manuele Migliardi,Antonio Rosato

doi:10.1074/jbc.m700695200

Abstract

ATP7A is a P-type ATPase involved in copper(I) homeostasis in humans. It possesses a long N-terminal cytosolic tail containing six domains that are individually folded and capable of binding one copper(I) ion each. We investigated the entire N-terminal tail (MNK1-6) in solution by NMR spectroscopy and addressed its interaction with copper(I) and with copper(I)-HAH1, the physiological partner of ATP7A. At copper(I)-HAH1:MNK1-6 ratios of up to 3:1, thus encompassing the range of protein ratios in vivo, both the first and fourth domain of the tail formed a metal-mediated adduct with HAH1 whereas the sixth domain was simultaneously able to partly remove copper(I) from HAH1. These processes are not dependent on one another. In particular, formation of the adducts is not necessary for copper(I) transfer from HAH1 to the sixth domain. The present data, together with available in vivo studies, suggest that the localization of ATP7A between the trans-Golgi network and the plasma membrane may be regulated by the accumulation of the adducts with HAH1, whereas the main role of domains 5 and 6 is to assist copper(I) translocation.

Highlights

Of copper from non-hepatic tissues and its absorption into circulation, and (ii) to deliver copper to the secretory pathway for incorporation into copper-dependent enzymes [9]
MNK is localized within the trans-Golgi network (TGN)2; copper stimulation results in the redistribution of MNK to the plasma membrane [3]
The analysis of 15N relaxation rates for the few signals observed from residues in the linker regions showed that the flexibility of these regions uncouples the motion in solution of the various domains with the exception of domains 5 and 6, which are separated by only six amino acids and behave more to a single rigid unit

Summary

Introduction

Of copper from non-hepatic tissues and its absorption into circulation, and (ii) to deliver copper to the secretory pathway for incorporation into copper-dependent enzymes [9]. Of copper(I) and of copper(I)-HAH1 with the N-terminal tail of MNK (MNK1– 6 hereafter) to obtain insights into the different behavior of the six metal-binding domains in the context of the entire cytosolic portion of the protein.

Results

Conclusion